Perinatal nicotine exposure-induced transgenerational asthma: Effects of reexposure in F1 gestation.

In a rat model, perinatal nicotine exposure results in an epigenetically driven multi- and trans-generationally transmitted asthmatic phenotype that tends to wane over successive generations. However, the effect of repeat nicotine exposure during the F1 (Filial 1) gestational period on the transmitted phenotype is unknown. Using a well-established rat model, we compared lung function, mesenchymal markers of airway reactivity, and global gonadal DNA methylation changes in F2 offspring in a sex-specific manner following perinatal exposure to nicotine in only the F0 gestation, in both F0 and F1 (F0/F1) gestations, and in neither (control group). Both F0 only and F0/F1 exposure groups showed an asthmatic phenotype, an effect that was more pronounced in the F0/F1 exposure group, especially in males. Testicular global DNA methylation increased, while ovarian global DNA methylation decreased in the F0/F1 exposed group. Since the offspring of smokers are more likely to smoke than the offspring of nonsmokers, this sets the stage for more severe asthma if both mother and grandmother had smoked during their pregnancies. Increased gonadal DNA methylation changes following nicotine reexposure in the F1 generation suggests that epigenetic mechanisms might well underlie the transgenerational inheritance of acquired phenotypic traits in general and nicotine-induced asthma in particular.

Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells.

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein-coupled receptor (GPCR) leading to PLCß activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Transferrin iron starvation therapy for lethal bacterial and fungal infections.

New strategies to treat antibiotic-resistant infections are urgently needed. We serendipitously discovered that stem cell conditioned media possessed broad antimicrobial properties. Biochemical, functional, and genetic assays confirmed that the antimicrobial effect was mediated by supra-physiological concentrations of transferrin. Human transferrin inhibited growth of gram-positive (Staphylococcus aureus), gram-negative (Acinetobacter baumannii), and fungal (Candida albicans) pathogens by sequestering iron and disrupting membrane potential. Serial passage in subtherapeutic transferrin concentrations resulted in no emergence of resistance. Infected mice treated with intravenous human transferrin had improved survival and reduced microbial burden. Finally, adjunctive transferrin reduced the emergence of rifampin-resistant mutants of S. aureus in infected mice treated with rifampin. Transferrin is a promising, novel antimicrobial agent that merits clinical investigation. These results provide proof of principle that bacterial infections can be treated in vivo by attacking host targets (ie, trace metal availability) rather than microbial targets.

The NOD/RIP2 pathway is essential for host defenses against Chlamydophila pneumoniae lung infection.

Here we investigated the role of the Nod/Rip2 pathway in host responses to Chlamydophila pneumoniae-induced pneumonia in mice. Rip2(-/-) mice infected with C. pneumoniae exhibited impaired iNOS expression and NO production, and delayed neutrophil recruitment to the lungs. Levels of IL-6 and IFN-gamma levels as well as KC and MIP-2 levels in bronchoalveolar lavage fluid (BALF) were significantly decreased in Rip2(-/-) mice compared to wild-type (WT) mice at day 3. Rip2(-/-) mice showed significant delay in bacterial clearance from the lungs and developed more severe and chronic lung inflammation that continued even on day 35 and led to increased mortality, whereas WT mice cleared the bacterial load, recovered from acute pneumonia, and survived. Both Nod1(-/-) and Nod2(-/-) mice also showed delayed bacterial clearance, suggesting that C. pneumoniae is recognized by both of these intracellular receptors. Bone marrow chimera experiments demonstrated that Rip2 in BM-derived cells rather than non-hematopoietic stromal cells played a key role in host responses in the lungs and clearance of C. pneumoniae. Furthermore, adoptive transfer of WT macrophages intratracheally was able to rescue the bacterial clearance defect in Rip2(-/-) mice. These results demonstrate that in addition to the TLR/MyD88 pathway, the Nod/Rip2 signaling pathway also plays a significant role in intracellular recognition, innate immune host responses, and ultimately has a decisive impact on clearance of C. pneumoniae from the lungs and survival of the infectious challenge.

Involvement of innate and adaptive immunity in a murine model of coronary arteritis mimicking Kawasaki disease.

Kawasaki disease (KD) is the most common cause of acquired cardiac disease and acute vasculitis in children in the developed world. Injection of a cell wall extract isolated from Lactobacillus casei (LCCWE) into mice causes a focal coronary arteritis that histopathologically mimics the coronary lesions observed in KD patients. In this study we used this model to investigate the participation of T cells, B cells, and dendritic cells (DC) in the development of coronary arteritis. RAG1(-/-), B cell(null), and wild-type (WT) mice were injected with a single dose of LCCWE (500 microg/mouse i.p.). None of the RAG1(-/-) mice developed coronary arteritis, whereas 70% of WT and 100% of B cell(null) mice developed coronary lesions, indicating that T cells were required for lesion formation. When splenocytes isolated from LCCWE-treated mice were restimulated with LCCWE, we observed significant IFN-gamma secretion in WT but not in RAG1(-/-) mice. Immunohistochemical staining showed F4/80(+) macrophages, activated MIDC-8(+) myeloid DCs (mDC), plasmacytoid DCs, and colocalization of CD3(+) T cells with mDCs in coronary artery lesions, suggesting an Ag-driven process. T cells but not B cells are required for LCCWE-induced coronary arteritis. Similar to human lesions, the coronary lesions contain macrophages, activated mDCs, and plaslmacytoid DCs all in close proximity to T cells, further strengthening the relevance of this mouse model to the immunopathology of coronary disease in KD. These studies are consistent with the interpretation that macrophages and DCs may collaborate with T cells in the pathological mechanisms of coronary arteritis.

Splenic lymphopenia in the endothelin receptor B-null mouse: implications for Hirschsprung associated enterocolitis.

PURPOSE: The aim of the study was to describe and characterize a novel small spleen phenotype with splenic lymphopenia in the Ednrb-null (Ednrb-/-) mouse with aganglionosis known to also develop enterocolitis. METHODS: We compared spleen weight as a percent of body weight from Ednrb+/+, Ednrb+/-, and Ednrb-/- mice to quantify our initial observation. Splenic microarchitecture of Ednrb+/+ and Ednrb-/- mice was assessed using both H and E staining and immunofluorescence staining for CD45R+ (B cells) and CD3+ (T cells) on tissue sections. To identify and quantify cell type, flow cytometry for CD19+ (mature B cells), CD4+ and CD8+ (T cells) was performed on the splenocytes of Ednrb+/+ and Ednrb-/- mice and compared with student's t test. A separate cohort of Ednrb+/+ and Ednrb-/- mice was killed and splenocytes were analyzed by flow cytometry, and proximal colon was histopathologically graded for enterocolitis. Spearman's rank correlations comparing total splenocyte and CD19+ cell counts with enterocolitis scores were performed. RESULTS: We found that the mean spleen weight expressed as a percent of body weight for Ednrb+/+ and Ednrb-/- mice was 0.72 and 0.25%, respectively (P < 0.001), at 25 days of age. In addition, the Ednrb-/- spleens also had markedly abnormal splenic microarchitecture with lymphopenia, and relative reduction of B cells compared to T cells. FACS of splenocytes revealed a 5 to 20-fold reduction in total cell number, CD19+, CD4+, and CD8+ of the Ednrb-/- mice compared to the Ednrb+/+ littermates (P < 0.01). We also found a strong inverse correlation of total spleen and CD19+ cell counts with histopathological enterocolitis scores (r (s) = -0.43, P = 0.02), showing that mice with reduced cell counts also had increased severity of enterocolitis. CONCLUSION: The small spleen immunophenotype in the Ednrb-/- mouse suggests that Ednrb-dependent signaling may be required for normal spleen development. These results raise the possibility that primary immune abnormalities may contribute at least in part to some enterocolitis. At present, our data suggest intriguing new potential explanations for HAEC in Hirschsprung patients.

Biomarkers for prediction of skeletal disease progression in mucopolysaccharidosis type I.

BACKGROUND: Orthopedic disease progresses in mucopolysaccharidosis type I (MPS I), even with approved therapies and remains a major factor in persistent suffering and disability. Novel therapies and accurate predictors of response are needed. The primary objective of this study was to identify surrogate biomarkers of future change in orthopedic disease. METHODS: As part of a 9-year observational study of MPS I, range-of-motion (ROM), height, pelvic radiographs were measured annually. Biomarkers in year 1 were compared to healthy controls. Linear regression tested for associations of change in biomarkers over the first year with change in long-term outcomes. RESULTS: MPS I participants (N = 19) were age 5 to 16 years and on average 6.9 ± 2.9 years post treatment initiation. Healthy controls (N = 51) were age 9 to 17 years. Plasma IL-1ß, TNF-α, osteocalcin, pyridinolines, and deoxypyridinolines were higher in MPS than controls. Within MPS, progression of hip dysplasia was present in 46% to 77%. A 1 pg/mL increase in IL-6 was associated with -22°/year change in ROM (-28 to -15; P < .001), a 20 nmol/mmol creatinine/year increase in urine PYD was associated with a -0.024 Z-score/year change in height Z-score (-0.043 to -0.005; P = .016), and a 20 nmol/mmol creatinine/year increase in urine PYD was associated with a -2.0%/year change in hip dysplasia measured by Reimers migration index (-3.8 to -0.1; P = .037). CONCLUSIONS: Inflammatory cytokines are high in MPS I. IL-6 and PYD were associated with progression in joint contracture, short stature, and hip dysplasia over time. Once validated, these biomarkers may prove useful for predicting response to treatment of skeletal disease in MPS I.

Chlamydia pneumoniae-induced foam cell formation requires MyD88-dependent and -independent signaling and is reciprocally modulated by liver X receptor activation.

Chlamydia pneumoniae is detected by macrophages and other APCs via TLRs and can exacerbate developing atherosclerotic lesions, but how that occurs is not known. Liver X receptors (LXRs) centrally control reverse cholesterol transport, but also negatively modulate TLR-mediated inflammatory pathways. We isolated peritoneal macrophages from wild-type, TLR2, TLR3, TLR4, TLR2/4, MyD88, TRIF, MyD88/TRIF, and IFN regulatory factor 3 (IRF3) KO mice, treated them with live or UV-killed C. pneumoniae in the presence or absence of oxidized LDL, then measured foam cell formation. In some experiments, the synthetic LXR agonist GW3965 was added to macrophages infected with C. pneumoniae in the presence of oxidized LDL. Both live and UV-killed C. pneumoniae induced IRF3 activation and promoted foam cell formation in wild-type macrophages, whereas the genetic absence of TLR2, TLR4, MyD88, TRIF, or IRF3, but not TLR3, significantly reduced foam cell formation. C. pneumoniae-induced foam cell formation was significantly reduced by the LXR agonist GW3965, which in turn inhibited C. pneumoniae-induced IRF3 activation, suggesting a bidirectional cross-talk. We conclude that C. pneumoniae facilitates foam cell formation via activation of both MyD88-dependent and MyD88-independent (i.e., TRIF-dependent and IRF3-dependent) pathways downstream of TLR2 and TLR4 signaling and that TLR3 is not involved in this process. This mechanism could at least partly explain why infection with C. pneumoniae accelerates the development of atherosclerotic plaque and lends support to the proposal that LXR agonists might prove clinically useful in suppressing atherogenesis.

TLR/MyD88 and liver X receptor alpha signaling pathways reciprocally control Chlamydia pneumoniae-induced acceleration of atherosclerosis.

Experimental and clinical studies link Chlamydia pneumoniae infection to atherogenesis and atherothrombotic events, but the underlying mechanisms are unclear. We tested the hypothesis that C. pneumoniae-induced acceleration of atherosclerosis in apolipoprotein E (ApoE)(-/-) mice is reciprocally modulated by activation of TLR-mediated innate immune and liver X receptor alpha (LXRalpha) signaling pathways. We infected ApoE(-/-) mice and ApoE(-/-) mice that also lacked TLR2, TLR4, MyD88, or LXRalpha intranasally with C. pneumoniae followed by feeding of a high fat diet for 4 mo. Mock-infected littermates served as controls. Atherosclerosis was assessed in aortic sinuses and in en face preparation of whole aorta. The numbers of activated dendritic cells (DCs) within plaques and the serum levels of cholesterol and proinflammatory cytokines were also measured. C. pneumoniae infection markedly accelerated atherosclerosis in ApoE-deficient mice that was associated with increased numbers of activated DCs in aortic sinus plaques and higher circulating levels of MCP-1, IL-12p40, IL-6, and TNF-alpha. In contrast, C. pneumoniae infection had only a minimal effect on atherosclerosis, accumulation of activated DCs in the sinus plaques, or circulating cytokine increases in ApoE(-/-) mice that were also deficient in TLR2, TLR4, or MyD88. However, C. pneumoniae-induced acceleration of atherosclerosis in ApoE(-/-) mice was further enhanced in ApoE(-/-)LXRalpha(-/-) double knockout mice and was accompanied by higher serum levels of IL-6 and TNF-alpha. We conclude that C. pneumoniae infection accelerates atherosclerosis in hypercholesterolemic mice predominantly through a TLR/MyD88-dependent mechanism and that LXRalpha appears to reciprocally modulate and reduce the proatherogenic effects of C. pneumoniae infection.

Chlamydial heat shock protein 60 induces acute pulmonary inflammation in mice via the Toll-like receptor 4- and MyD88-dependent pathway.

Heat shock protein 60 derived from Chlamydia pneumoniae (cHSP60) activates Toll-like receptor 4 (TLR4) signaling through the MyD88 pathway in vitro, but it is not known how cHSP60 contributes to C. pneumoniae-induced lung inflammation. We treated wild-type (WT), TLR2(-/-), TLR4(-/-), or MyD88(-/-) mice intratracheally (i.t.) with recombinant cHSP60 (50 microg), UV-killed C. pneumoniae (UVCP; 5 x 10(6) inclusion-forming units/mouse), lipopolysaccharide (2 microg), or phosphate-buffered saline (PBS) and sacrificed mice 24 h later. Bronchoalveolar lavage (BAL) was obtained to measure cell counts and cytokine levels, lungs were analyzed for histopathology, and lung homogenate chemokine concentrations were determined. Bone marrow-derived dendritic cells (BMDDCs) were generated and stimulated with live C. pneumoniae (multiplicity of infection [MOI], 5), UVCP (MOI, 5), or cHSP60 for 24 h, and the expression of costimulatory molecules (CD80 and CD86) was measured by fluorescence-activated cell sorting. cHSP60 induced acute lung inflammation with the same intensity as that of UVCP-induced inflammation in WT mice but not in TLR4(-/-) or MyD88(-/-) mice. cHSP60- and UVCP-induced lung inflammation was associated with increased numbers of cells in BAL, increased neutrophil recruitment, and elevated BAL interleukin-6 (IL-6) levels. Both cHSP60 and UVCP induced IL-6 release and CD80 and CD86 expression in WT cells but not in MyD88(-/-) BMDDCs. cHSP60 stimulated DC activation in a TLR4- and MyD88-dependent manner with an intensity similar to that induced by UVCP. These data suggest that cHSP60 promotes lung inflammation and DC activation via TLR4 and MyD88 and therefore may play a significant role in the pathogenesis of C. pneumoniae-induced chronic inflammatory lung diseases.

Innate immune responses during respiratory tract infection with a bacterial pathogen induce allergic airway sensitization.

BACKGROUND: The original hygiene hypothesis predicts that infections should protect against asthma but does not account for increasing evidence that certain infections might also promote asthma development. A mechanistic reconciliation of these findings has not yet emerged. In particular, the role of innate immunity in this context is unclear. OBJECTIVE: We sought to test whether bacterial respiratory tract infection causes airway sensitization toward an antigen encountered in parallel and to elucidate the contribution of innate immune responses. METHODS: Mice were infected with different doses of Chlamydia pneumoniae, followed by exposure to human serum albumin (HSA) and challenge with HSA 2 weeks later. Airway inflammation, immunoglobulins, and lymph node cytokines were assessed. Furthermore, adoptive transfer of dendritic cells (DCs) and depletion of regulatory T (Treg) cells was performed. RESULTS: C pneumoniae-induced lung inflammation triggered sensitization toward HSA, resulting in eosinophilic airway inflammation after HSA challenge. Airway sensitization depended on the severity and timing of infection: low-dose infection and antigen exposure within 5 days of infection induced allergic sensitization, whereas high-dose infection or antigen exposure 10 days after infection did not. Temporal and dose-related effects reflected DC activation and could be reproduced by means of adoptive transfer of HSA-pulsed lung DCs from infected mice. MyD88 deficiency in DCs abolished antigen sensitization, and depletion of Treg cells prolonged the time window in which sensitization could occur. CONCLUSIONS: We conclude that moderate, but not severe, pulmonary bacterial infection can induce allergic sensitization to inert inhaled antigens through a mechanism that requires MyD88-dependent DC activation and is controlled by Treg cells.

Molecular, endocrine, and genetic mechanisms of arterial calcification.

Pathologists have recognized arterial calcification for over a century. Recent years have witnessed a strong resurgence of interest in atherosclerotic plaque calcification because it: 1) can be easily detected noninvasively; 2) closely correlates with the amount of atherosclerotic plaque; 3) serves as a surrogate measure for atherosclerosis, allowing preclinical detection of the disease; and 4) is associated with heightened risk of adverse cardiovascular events. There are two major types of calcification in arteries: calcification of the media tunica layer (sometimes called Mönckeberg's sclerosis), and calcification within subdomains of atherosclerotic plaque within the intimal layer of the artery. There are important similarities and differences between these two entities. Of particular interest are increasing parallels between cellular and molecular features of arterial calcification and bone biology, and this has led to accelerating interest in understanding how and why bone-like mineral deposits may form in arteries. Here, we review the two major pathological types of arterial calcification, the proposed models of calcification, and endocrine and genetic determinants that affect arterial calcification. In addition, we highlight areas requiring further investigation.

A Humoral Immune Response Alters the Distribution of Enzyme Replacement Therapy in Murine Mucopolysaccharidosis Type I.

Antibodies against recombinant proteins can significantly reduce their effectiveness in unanticipated ways. We evaluated the humoral response of mice with the lysosomal storage disease mucopolysaccharidosis type I treated with weekly intravenous recombinant human alpha-l-iduronidase (rhIDU). Unlike patients, the majority of whom develop antibodies to recombinant human alpha-l-iduronidase, only approximately half of the treated mice developed antibodies against recombinant human alpha-l-iduronidase and levels were low. Serum from antibody-positive mice inhibited uptake of recombinant human alpha-l-iduronidase into human fibroblasts by partial inhibition compared to control serum. Tissue and cellular distributions of rhIDU were altered in antibody-positive mice compared to either antibody-negative or naive mice, with significantly less recombinant human alpha-l-iduronidase activity in the heart and kidney in antibody-positive mice. In the liver, recombinant human alpha-l-iduronidase was preferentially found in sinusoidal cells rather than in hepatocytes in antibody-positive mice. Antibodies against recombinant human alpha-l-iduronidase enhanced uptake of recombinant human alpha-l-iduronidase into macrophages obtained from MPS I mice. Collectively, these results imply that a humoral immune response against a therapeutic protein can shift its distribution preferentially into macrophage-lineage cells, causing decreased availability of the protein to the cells that are its therapeutic targets.

TB, or not TB: that is the question -- does TLR signaling hold the answer?

Innate immunity critically depends on signaling by Toll-like receptors (TLRs) that rely heavily on an intracellular adapter protein called myeloid differentiation factor 88 (MyD88). Adaptive immune defenses are generally thought to be orchestrated by innate immune responses and so should require intact TLR-MyD88 signaling pathways. But a surprising new study in MyD88-null mice infected with Mycobacterium tuberculosis challenges this view and instead suggests that MyD88 may not be absolutely required for a normal adaptive immune response.

Diabetes Exacerbates Infection via Hyperinflammation by Signaling through TLR4 and RAGE.

For more than a century, diabetic patients have been considered immunosuppressed due to defects in phagocytosis and microbial killing. We confirmed that diabetic mice were hypersusceptible to bacteremia caused by Gram-negative bacteria (GNB), dying at inocula nonlethal to nondiabetic mice. Contrary to the pervasive paradigm that diabetes impedes phagocytic function, the bacterial burden was no greater in diabetic mice despite excess mortality. However, diabetic mice did exhibit dramatically increased levels of proinflammatory cytokines in response to GNB infections, and immunosuppressing these cytokines with dexamethasone restored their resistance to infection, both of which are consistent with excess inflammation. Furthermore, disruption of the receptor for advanced glycation end products (RAGE), which is stimulated by heightened levels of AGEs in diabetic hosts, protected diabetic but not nondiabetic mice from GNB infection. Thus, rather than immunosuppression, diabetes drives lethal hyperinflammation in response to GNB by signaling through RAGE. As such, interventions to improve the outcomes from GNB infections should seek to suppress the immune response in diabetic hosts.IMPORTANCE Physicians and scientists have subscribed to the dogma that diabetes predisposes the host to worse outcomes from infections because it suppresses the immune system. This understanding was based largely on ex vivo studies of blood from patients and animals with diabetes. However, we have found that the opposite is true and worse outcomes from infection are caused by overstimulation of the immune system in response to bacteria. This overreaction occurs by simultaneous ligation of two host receptors: TLR4 and RAGE. Both signal via a common downstream messenger, MyD88, triggering hyperinflammation. In summary, contrary to hundred-year-old postulations about immune suppression in diabetic hosts, we find that diabetes instead predisposes to more severe infections because of additional inflammatory output through dual activation of MyD88 by not only TLR4 but also RAGE. It is the activation of RAGE during GNB infections in those with diabetes that accounts for their heightened susceptibility to infection compared to nondiabetic hosts.

TLR2 and MyD88 contribute to Lactobacillus casei extract-induced focal coronary arteritis in a mouse model of Kawasaki disease.

BACKGROUND: Kawasaki disease is the most common cause of acquired cardiac disease and acute vasculitis in children, targets the coronary arteries, and can occasionally be fatal. The pathogenesis and the molecular mechanisms remain unknown. After injection of Lactobacillus casei cell-wall extract (LCCWE), mice develop a focal coronary arteritis that histopathologically resembles Kawasaki disease, but the mechanism remains unclear. Here, we tested the hypothesis that signaling by Toll-like receptors (TLRs) through their key downstream adaptor molecule myeloid differentiation factor 88 (MyD88) is required for the cellular activation and coronary arteritis produced by LCCWE. METHODS AND RESULTS: Bone marrow-derived macrophages from TLR2- or MyD88-deficient mice were unresponsive to LCCWE-induced stimulation. In contrast, macrophages obtained from TLR4-deficient mice produced the same amount of interleukin-6 as macrophages from wild-type mice after stimulation with LCCWE. Intraperitoneal injection of LCCWE produced severe focal coronary arteritis in TLR4(-/-) and C57BL/6 control mice but not in TLR2(-/-) or MyD88(-/-) mice. Collectively, these results indicate that LCCWE is a potent inducer of nuclear factor-kappaB via TLR2 but not TLR4 and that this activation proceeds via the MyD88-dependent signaling pathway. In vivo studies suggest that TLR2(-/-) mice are protected from LCCWE-induced coronary arteritis and that this protection is mediated through the adaptor molecule MyD88. CONCLUSIONS: Our results provide important insights into the molecular signaling in this mouse model of coronary arteritis. We show here that LCCWE-induced coronary arteritis is dependent on intact TLR2 and MyD88 signaling.

Ubiquitination and de-ubiquitination: role in regulation of signaling by Toll-like receptors.

Signaling by Toll-like receptors (TLRs) has attracted accelerating attention over the past decade because of the central role of TLR signaling in both innate and adaptive immunity. In addition, TLR signaling is now increasingly implicated in a remarkably wide range of diseases that are either caused, or accompanied, by dysregulated inflammation. Much has been learned about the basic signaling framework and participants, as well as how signaling is turned off and fine-tuned. Here, we summarize key aspects of TLR signaling, focusing on interaction with the anti-inflammatory TGF-beta signaling network. We propose that ubiquitination and de-ubiquitination of TLR pathway components may be a mechanism by which predominantly anti-inflammatory input is integrated into the host response to fine-tune inflammation in accordance with the needs of host defenses.

Increased expression of macrophage colony-stimulating factor after coronary artery balloon injury is inhibited by intracoronary brachytherapy.

BACKGROUND: The mechanisms underlying the reduced neointimal proliferation (NP) by intracoronary brachytherapy (ICBT) are unknown. We hypothesized that ICBT inhibits NP by reducing expression of macrophage colony-stimulating factor (M-CSF). METHODS AND RESULTS: Thirty coronary arteries from 10 pigs were divided into 3 groups of 10 each: control (C), balloon injury (BI), and BI followed by ICBT (16 Gy at 0.5-mm tissue depth with a (32)P balloon system). Pigs were killed at 24 hours (n=3) and at 7 (n=4) and 14 (n=3) days. Expression of M-CSF was assessed by Western blot, ELISA, and quantitative immunostaining. Persistently increased levels of M-CSF after BI (to 1.4+/-0.2 nmol/L [ELISA] and 29.4+/-4.9% of cross-sectional area stained [immunocytochemistry]; P< 0.001 versus control for both) were observed in the injured arteries. Treatment of BI arteries with ICBT reduced M-CSF expression compared with BI alone (to 0.7+/-0.1 nmol/L [ELISA] and 13.5+/-2.9% of cross-sectional area stained [immunocytochemistry]; P<0.001 versus BI and P=NS versus control for both) and remained similar to control M-CSF expression for the 14-day study period. Neointimal thickness increased after BI (to 4.8+/-2.9 mm(2); P<0.001 versus control), but this was reduced by ICBT (1.4+/-0.4 mm(2); P<0.001 versus BI). CONCLUSIONS: In porcine coronary arteries, BI is associated with increased expression of M-CSF and NP, but neither occurs after ICBT. The beneficial effects of ICBT on NP involve inhibition of M-CSF expression.

Increased expression of membrane type 3-matrix metalloproteinase in human atherosclerotic plaque: role of activated macrophages and inflammatory cytokines.

BACKGROUND: Matrix metalloproteinases (MMPs) are thought to play a prominent role in atherogenesis and destabilization of plaque. Pericellularly localized membrane-type (MT)-MMPs activate secreted MMPs. We investigated the hypothesis that MT3-MMP is expressed in human atherosclerotic plaques and is regulated by locally produced inflammatory cytokines and oxidized low-density lipoprotein (Ox-LDL). METHODS AND RESULTS: Expression and cellular localization of MT3-MMP in normal and atherosclerotic human coronary arteries were examined using specific antibodies. Abundant MT3-MMP expression was noted in medial smooth muscle cells (SMCs) of normal arteries. In atherosclerotic arteries, MT3-MMP expression was observed within complex plaques and colocalized with SMCs and macrophages (Mphi). Cultured human monocyte-derived Mphi constitutively expressed MT3-MMP mRNA and proteolytically active protein, as demonstrated by mRNA analyses, immunoblotting, and gelatin zymography, respectively. Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor caused dose- and time-dependent increases in steady-state levels of MT3-MMP mRNA in cultured Mphi. This correlated with a 2- to 4-fold increase in levels of MT3-MMP immunoreactive protein and enzymatic activity in Mphi membranes. Confocal microscopy and flow cytometry confirmed induction and spatial distribution of MT3-MMP protein from intracellular domains to the Mphi plasma membrane by Ox-LDL, tumor necrosis factor-alpha, or macrophage colony-stimulating factor. CONCLUSIONS: MT3-MMP is expressed by SMCs and Mphi in human atherosclerotic plaques. Proinflammatory molecules cause a progressive increase in the expression of MT3-MMP in cultured Mphi. Our results suggest a mechanism by which inflammatory molecules could promote Mphi-mediated degradation of extracellular matrix and thereby contribute to plaque destabilization.

Calcification of the coronary arteries in the absence of atherosclerotic plaque.

Arterial calcification in the coronary arteries frequently indicates concomitant atherosclerotic plaque but can be present in the medial layers with no evidence of plaque. Calcification of the medial layer of arteries is seen most often in the peripheral arteries but also is widely recognized In the coronary arteries. We describe 2 patients who had marked medial and intimal calcification of the coronary arteries with little or no accompanying atherosclerosis.