Plasma proteome of growing tumors.

Early detection of cancer is vital for the best chance of successful treatment, but half of all cancers are diagnosed at an advanced stage. A simple and reliable blood screening test applied routinely would therefore address a major unmet medical need. To gain insight into the value of protein biomarkers in early detection and stratification of cancer we determined the time course of changes in the plasma proteome of mice carrying transplanted human lung, breast, colon, or ovarian tumors. For protein measurements we used an aptamer-based assay which simultaneously measures ~ 5000 proteins. Along with tumor lineage-specific biomarkers, we also found 15 markers shared among all cancer types that included the energy metabolism enzymes glyceraldehyde-3-phosphate dehydrogenase, glucose-6-phophate isomerase and dihydrolipoyl dehydrogenase as well as several important biomarkers for maintaining protein, lipid, nucleotide, or carbohydrate balance such as tryptophanyl t-RNA synthetase and nucleoside diphosphate kinase. Using significantly altered proteins in the tumor bearing mice, we developed models to stratify tumor types and to estimate the minimum detectable tumor volume. Finally, we identified significantly enriched common and unique biological pathways among the eight tumor cell lines tested.

Pseudomonas aeruginosa LecB suppresses immune responses by inhibiting transendothelial migration.

Pseudomonas aeruginosa is a Gram-negative bacterium causing morbidity and mortality in immuno-compromised humans. It produces a lectin, LecB, that is considered a major virulence factor, however, its impact on the immune system remains incompletely understood. Here we show that LecB binds to endothelial cells in human skin and mice and disrupts the transendothelial passage of leukocytes in vitro. It impairs the migration of dendritic cells into the paracortex of lymph nodes leading to a reduced antigen-specific T cell response. Under the effect of the lectin, endothelial cells undergo profound cellular changes resulting in endocytosis and degradation of the junctional protein VE-cadherin, formation of an actin rim, and arrested cell motility. This likely negatively impacts the capacity of endothelial cells to respond to extracellular stimuli and to generate the intercellular gaps for allowing leukocyte diapedesis. A LecB inhibitor can restore dendritic cell migration and T cell activation, underlining the importance of LecB antagonism to reactivate the immune response against P. aeruginosa infection.

Lymphoid stromal cells - potential implications for the pathogenesis of CVID.

Non-hematopoietic lymphoid stromal cells (LSC) maintain lymph node architecture and form niches allowing the migration, activation, and survival of immune cells. Depending on their localization in the lymph node, these cells display heterogeneous properties and secrete various factors supporting the different activities of the adaptive immune response. LSCs participate in the transport of antigen from the afferent lymph as well as in its delivery into the T and B cell zones and organize cell migration via niche-specific chemokines. While marginal reticular cells (MRC) are equipped for initial B-cell priming and T zone reticular cells (TRC) provide the matrix for T cell-dendritic cell interactions within the paracortex, germinal centers (GC) only form when both T- and B cells successfully interact at the T-B border and migrate within the B-cell follicle containing the follicular dendritic cell (FDC) network. Unlike most other LSCs, FDCs are capable of presenting antigen via complement receptors to B cells, which then differentiate within this niche and in proximity to T follicular helper (TFH) cells into memory and plasma cells. LSCs are also implicated in maintenance of peripheral immune tolerance. In mice, TRCs induce the alternative induction of regulatory T cells instead of TFH cells by presenting tissue-restricted self-antigens to naïve CD4 T cells via MHC-II expression. This review explores potential implications of our current knowledge of LSC populations regarding the pathogenesis of humoral immunodeficiency and autoimmunity in patients with autoimmune disorders or common variable immunodeficiency (CVID), the most common form of primary immunodeficiency in humans.

Searching for DNA methylation in patients triple-negative breast cancer: a liquid biopsy approach.

INTRODUCTION: Liquid biopsies are proving to have diagnostic and prognostic value in many different cancers, and in breast cancer they have the potential to improve outcomes by providing valuable information throughout a patient's cancer journey. However, patients with triple negative breast cancer (TNBC) have received little benefit from such liquid biopsies due to underlying limitations in the discovery and utility of robust biomarkers. Here, we examine the development of DNA methylation-based liquid biopsy assays for breast cancer and how they pertain to TNBC. AREAS COVERED: We conducted a systematic review of liquid biopsy assays for breast cancer and analyzed their relevance in TNBC. We show that the utility of DNA mutation-based assays is poor for TNBC due to the low mutational frequencies across the genome in this subtype. We offer a detailed review of mDETECT - a liquid biopsy specifically designed for assessing tumor burden in TNBC patients. EXPERT OPINION: DNA methylation are foundational and robust events that occur in cancer evolution and may differentiate almost all forms of cancer, including TNBC. Longitudinal patient monitoring using DNA methylation-based liquid biopsies offers great potential for improving the detection and management of TNBC.

Engineering Novel 3D Models to Recreate High-Grade Osteosarcoma and its Immune and Extracellular Matrix Microenvironment.

Osteosarcoma (OS) is the most common primary bone cancer, where the overall 5-year surviving rate is below 20% in resistant forms. Accelerating cures for those poor outcome patients remains a challenge. Nevertheless, several studies of agents targeting abnormal cancerous pathways have yielded disappointing results when translated into clinic because of the lack of accurate OS preclinical modeling. So, any effort to design preclinical drug testing may consider all inter-, intra-, and extra-tumoral heterogeneities throughout models mimicking extracellular and immune microenvironment. Therefore, the bioengineering of patient-derived models reproducing the OS heterogeneity, the interaction with tumor-associated macrophages (TAMs), and the modulation of oxygen concentrations additionally to recreation of bone scaffold is proposed here. Eight 2D preclinical models mimicking several OS clinical situations and their TAMs in hypoxic conditions are developed first and, subsequently, the paired 3D models faithfully preserving histological and biological characteristics are generated. It is possible to shape reproducibly M2-like macrophages cultured with all OS patient-derived cell lines in both dimensions. The final 3D models pooling all heterogeneity features are providing accurate proliferation and migration data to understand the mechanisms involved in OS and immune cells/biomatrix interactions and sustained such that engineered 3D preclinical systems will improve personalized medicine.

CD169+ macrophages in lymph node and spleen critically depend on dual RANK and LTbetaR signaling.

CD169+ macrophages reside in lymph node (LN) and spleen and play an important role in the immune defense against pathogens. As resident macrophages, they are responsive to environmental cues to shape their tissue-specific identity. We have previously shown that LN CD169+ macrophages require RANKL for formation of their niche and their differentiation. Here, we demonstrate that they are also dependent on direct lymphotoxin beta (LTß) receptor (R) signaling. In the absence or the reduced expression of either RANK or LTßR, their differentiation is perturbed, generating myeloid cells expressing SIGN-R1 in LNs. Conditions of combined haploinsufficiencies of RANK and LTßR revealed that both receptors contribute equally to LN CD169+ macrophage differentiation. In the spleen, the Cd169-directed ablation of either receptor results in a selective loss of marginal metallophilic macrophages (MMMs). Using a RANKL reporter mouse, we identify splenic marginal zone stromal cells as a source of RANKL and demonstrate that it participates in MMM differentiation. The loss of MMMs had no effect on the splenic B cell compartments but compromised viral capture and the expansion of virus-specific CD8+ T cells. Taken together, the data provide evidence that CD169+ macrophage differentiation in LN and spleen requires dual signals from LTßR and RANK with implications for the immune response.

Current Clinical and Pre-Clinical Imaging Approaches to Study the Cancer-Associated Immune System.

In the light of the success and the expected growth of its arsenal, immuno-therapy may become the standard neoadjuvant procedure for many cancers in the near future. However, aspects such as the identity, organization and the activation status of the peri- and intra-tumoral immune cells would represent important elements to weigh in the decision for the appropriate treatment. While important progress in non-invasive imaging of immune cells has been made over the last decades, it falls yet short of entering the clinics, let alone becoming a standard procedure. Here, we provide an overview of the different intra-vital imaging approaches in the clinics and in pre-clinical settings and discuss their benefits and drawbacks for assessing the activity of the immune system, globally and on a cellular level. Stimulated by further research, the future is likely to see many technological advances both on signal detection and emission as well as image specificity and resolution to tackle current hurdles. We anticipate that the ability to precisely determine an immune stage of cancer will capture the attention of the oncologist and will create a change in paradigm for cancer therapy.

Abnormal hearing patterns are not associated with endothelium-dependent vasodilation and carotid intima-media thickness: The Framingham Heart Study.

INTRODUCTION: Prior data suggest associations between hearing loss, cardiovascular (CV) risk factors, and CV disease. Whether specific hearing loss patterns, including a strial pattern associated with inner ear vascular disease, are associated with systemic endothelial dysfunction and carotid intima-media thickness (IMT) remains unclear. METHODS: We evaluated participants without prevalent CVD in the Framingham Offspring Study who underwent formal audiogram testing and brachial and carotid artery ultrasounds. Audiograms were categorized as normal or as belonging to one of four abnormal patterns: cochlear-conductive, low-sloping, sensorineural, or strial. Endothelial function as measured by brachial artery flow-mediated dilation (FMDmm and FMD%). Internal and common intima-media thicknesses (icIMT and ccIMT, respectively) were compared between audiogram patterns. RESULTS: We studied 1672 participants (mean age 59 years, 57.6% women). The prevalence of each hearing pattern was as follows: 43.7% normal; 20.3% cochlear-conductive; 20.3% sensorineural; 7.7% low-sloping; and 8.0% strial. Strial pattern hearing loss was nearly twice as prevalent (p = 0.001) in those in the highest quartile of ccIMT and nearly 50% higher in those in the highest icIMT quartile (p = 0.04). There were no statistically significant differences between the prevalence of the strial pattern comparing the lowest quartiles of FMDmm and FMD% with the upper three quartiles. Age- and sex-adjusted linear regression models did not show significant associations between the vascular measures and hearing patterns. CONCLUSION: Abnormal hearing patterns were not significantly associated with impaired brachial FMD and increased carotid IMT after adjusting for age and sex effects, which may reflect age and sex-related distributional differences based on hearing loss pattern.

A DNA methylation-based liquid biopsy for triple-negative breast cancer.

Here, we present a next-generation sequencing (NGS) methylation-based blood test called methylation DETEction of Circulating Tumour DNA (mDETECT) designed for the optimal detection and monitoring of metastatic triple-negative breast cancer (TNBC). Based on a highly multiplexed targeted sequencing approach, this assay incorporates features that offer superior performance and included 53 amplicons from 47 regions. Analysis of a previously characterised cohort of women with metastatic TNBC with limited quantities of plasma (<2 ml) produced an AUC of 0.92 for detection of a tumour with a sensitivity of 76% for a specificity of 100%. mDETECTTNBC was quantitative and showed superior performance to an NGS TP53 mutation-based test carried out on the same patients and to the conventional CA15-3 biomarker. mDETECT also functioned well in serum samples from metastatic TNBC patients where it produced an AUC of 0.97 for detection of a tumour with a sensitivity of 93% for a specificity of 100%. An assay for BRCA1 promoter methylation was also incorporated into the mDETECT assay and functioned well but its clinical significance is currently unclear. Clonal Hematopoiesis of Indeterminate Potential was investigated as a source of background in control subjects but was not seen to be significant, though a link to adiposity may be relevant. The mDETECTTNBC assay is a liquid biopsy able to quantitatively detect all TNBC cancers and has the potential to improve the management of patients with this disease.

Vitamin D3-elicited CD14+ human skin dendritic cells promote thymic stromal lymphopoietin-independent type 2 T-helper responses.

BACKGROUND: Immune modulation by vitamin D3 through dendritic cells (DCs) remains controversial. Human DCs exposed in vitro counteract type-1 T-helper (Th1) differentiation and induce regulatory T cells. However, cutaneous application on mice promotes Th2-driven inflammation resembling atopic dermatitis and relying on thymic stromal lymphopoietin (TSLP) from keratinocytes and T-cell orientation by TSLP-stimulated skin DCs. We studied the effects of vitamin D3 in human skin, focusing on TSLP production and the role of skin DCs in T-cell differentiation. METHODS: Human healthy skin explants were exposed in vitro to vitamin D3 analogs. Migrating DCs were analyzed and TSLP quantified in the supernatant. Allogeneic naïve CD4+ T cells were cocultured with DCs to assess their proliferation and cytokine production. RESULTS: Vitamin D3 induced skin DCs to differentiate Th2 cells producing IL-4 and IL-13. Vitamin D3 triggered TSLP release in ~30% of skin explants, correlating with IL-13 detection in Th2 cells. In these donors, blocking TSLP receptor during skin explant cultures abrogated IL-13 production, yet IL-4+ Th2 cells were unaffected. Among skin DCs emerged CD14+ cells that had responded directly to vitamin D3 and differed from classical CD14+ dermal emigrants. Vitamin D3-elicited CD14+ DCs sufficed to promote IL-4+ Th2 cells in a TSLP-independent manner. CONCLUSION: Vitamin D3, despite inducing TSLP in some donors, had a direct influence on skin DCs, affecting their phenotype and ability to drive Th2 responses independently of TSLP. Our findings pave the way toward in vitro systems that accurately model human cutaneous Th2 responses, notably involved in atopic dermatitis.

Tenascin-C Orchestrates an Immune-Suppressive Tumor Microenvironment in Oral Squamous Cell Carcinoma.

Inherent immune suppression represents a major challenge in the treatment of human cancer. The extracellular matrix molecule tenascin-C promotes cancer by multiple mechanisms, yet the roles of tenascin-C in tumor immunity are incompletely understood. Using a 4NQO-induced oral squamous cell carcinoma (OSCC) model with abundant and absent tenascin-C, we demonstrated that tenascin-C enforced an immune-suppressive lymphoid stroma via CCL21/CCR7 signaling, leading to increased metastatic tumors. Through TLR4, tenascin-C increased expression of CCR7 in CD11c+ myeloid cells. By inducing CCL21 in lymphatic endothelial cells via integrin α9ß1 and binding to CCL21, tenascin-C immobilized CD11c+ cells in the stroma. Inversion of the lymph node-to-tumor CCL21 gradient, recruitment of T regulatory cells, high expression of anti-inflammatory cytokines, and matrisomal components were hallmarks of the tenascin-C-instructed lymphoid stroma. Ablation of tenascin-C or CCR7 blockade inhibited the lymphoid immune-suppressive stromal properties, reducing tumor growth, progression, and metastasis. Thus, targeting CCR7 could be relevant in human head and neck tumors, as high tenascin-C expression and an immune-suppressive stroma correlate to poor patient survival.

Development and initial clinical correlation of a DNA methylation-based blood test for prostate cancer.

BACKGROUND: One of the principle limitations for more precise management of advanced prostate cancer is the lack of accurate biomarkers allowing estimation of tumor burden, ongoing assessment of progression, and response to treatment. Although prostate-specific antigen (PSA) performs modestly, nonsecreting cancers including those with early castrate-resistance warrant investigation of other predictive biomarkers. The objectives of these studies were to develop and perform initial validation of a circulating tumor DNA (ctDNA) methylation assay. METHODS: Methylation DETection of Circulating Tumor DNA (mDETECT) is a highly multiplexed targeted sequencing DNA methylation-based ctDNA blood test that captures the vast majority of prostate cancer phenotypes due to a careful development process that ensures that each probe region is methylated in at least 50% of all methylation-based subtypes and is not methylated in normal tissues. Next-generation sequencing of targeted polymerase chain reaction (PCR) products whose amplification is biased towards methylated DNA ensures the specificity of the assay by identifying multiple tumor-specific methylated CpG residues in each read. RESULTS: The final test is comprised of 46 PCR probes to 40 regions. It is relatively resistant to contaminating normal DNA and as a result functions in both serum and plasma samples. The assay was initially validated in a variety of prostate cancer cell lines to ensure specificity. Using a small number of longitudinal samples from prostate cancer patients initiating androgen deprivation therapy, the ability of mDETECT to track tumor burden was assessed compared with PSA. The mDETECT test signal generally paralleled that of PSA increasing and decreasing commensurate with tumor evolution in these patients. In two cases it appeared to anticipate clinical progression by a number of months compared to PSA and in a PSA nonproducing case, it was able to track tumor progression. CONCLUSIONS: mDETECT offers a promising tool for the assessment of prostate cancer burden based on the sensitive detection of prostate-specific ctDNA and requires further validation.

BRCAness, SLFN11, and RB1 loss predict response to topoisomerase I inhibitors in triple-negative breast cancers.

Topoisomerase I (TOP1) inhibitors trap TOP1 cleavage complexes resulting in DNA double-strand breaks (DSBs) during replication, which are repaired by homologous recombination (HR). Triple-negative breast cancer (TNBC) could be eligible for TOP1 inhibitors given the considerable proportion of tumors with a defect in HR-mediated repair (BRCAness). The TOP1 inhibitor irinotecan was tested in 40 patient-derived xenografts (PDXs) of TNBC. BRCAness was determined with a single-nucleotide polymorphism (SNP) assay, and expression of Schlafen family member 11 (SLFN11) and retinoblastoma transcriptional corepressor 1 (RB1) was evaluated by real-time polymerase chain reaction (RT-PCR) and immunohistochemistry analyses. In addition, the combination of irinotecan and the ataxia telangiectasia and Rad3-related protein (ATR) inhibitor VE-822 was tested in SLFN11-negative PDXs, and two clinical non-camptothecin TOP1 inhibitors (LMP400 and LMP776) were tested. Thirty-eight percent of the TNBC models responded to irinotecan. BRCAness combined with high SLFN11 expression and RB1 loss identified highly sensitive tumors, consistent with the notion that deficiencies in cell cycle checkpoints and DNA repair result in high sensitivity to TOP1 inhibitors. Treatment by the ATR inhibitor VE-822 increased sensitivity to irinotecan in SLFN11-negative PDXs and abolished irinotecan-induced phosphorylation of checkpoint kinase 1 (CHK1). LMP400 (indotecan) and LMP776 (indimitecan) showed high antitumor activity in BRCA1-mutated or BRCAness-positive PDXs. Last, low SLFN11 expression was associated with poor survival in 250 patients with TNBC treated with anthracycline-based chemotherapy. In conclusion, a substantial proportion of TNBC respond to irinotecan. BRCAness, high SLFN11 expression, and RB1 loss are highly predictive of response to irinotecan and the clinical indenoisoquinoline TOP1 inhibitors.

Region-specific glucocorticoid receptor promoter methylation has both positive and negative prognostic value in patients with estrogen receptor-positive breast cancer.

BACKGROUND: The glucocorticoid receptor (NR3C1, GR) is frequently downregulated in breast tumors, and evidence suggests it acts as a tumor suppressor in estrogen receptor-positive (ER+) breast cancer. We previously found that methylation of the GR promoter CpG island represses gene expression and occurs in ER+ breast tumors. In this study, the prognostic and predictive value of GR methylation was examined in ER+ patients from the CCTG MA.12 clinical trial of tamoxifen versus placebo in women with early breast cancer. METHODS: We developed a targeted multiplex bisulfite next-generation sequencing assay to detect methylation at multiple GR promoter regions in DNA from formalin-fixed paraffin-embedded (FFPE) samples. Following validation in a small cohort of breast tumors, ER+ FFPE tumor samples from MA.12 (n = 208) were tested. Survival analyses evaluated the impact of GR promoter methylation on patient overall survival (OS) and disease-free survival (DFS). RESULTS: An analysis of TCGA data found that GR methylation is prevalent in ER+ tumors and is associated with decreased gene expression and analysis of public microarray data (KM Plotter) linked decreased GR expression to a poor outcome. In MA.12, two GR promoter regions (U and C) each had prognostic value, but with opposite effects on the outcome. U methylation was associated with poor OS (HR = 1.79, P = 0.041) whereas C methylation was associated with better OS (HR = 0.40, P = 0.040) and DFS (HR = 0.49, P = 0.037). The classification of patients based on the methylation status of the two regions was prognostic for OS (P = 0.006) and DFS (P = 0.041) and revealed a group of patients (U methylated, C unmethylated) with very poor outcomes. Placebo-treated patients in this high-risk group had worse OS (HR = 2.86, P = 0.002) and DFS (HR = 2.09, P = 0.014) compared to the rest of the cohort. CONCLUSION: Region-specific GR promoter methylation was an independent prognostic marker for patient survival and identified a subset of patients with poor prognosis, particularly without tamoxifen treatment. These findings provide a foundation for future studies into GR methylation as a promising prognostic biomarker in ER+ breast cancer.

Genetically-achieved disturbances to the expression levels of TNFSF11 receptors modulate the effects of zoledronic acid on growing mouse skeletons.

Zoledronic acid (ZOL), a nitrogen bisphosphonate (N-BP), is currently used to treat and control pediatric osteolytic diseases. Variations in the intensity of the effects and side effects of N-BPs have been reported with no clear explanations regarding their origins. We wonder if such variations could be associated with different levels of RANKL signaling activity in growing bone during and after the treatment with N-BPs. To answer this question, ZOL was injected into neonate C57BL/6J mice with different genetically-determined RANKL signaling activity levels (Opg+/+\RankTg-, Opg+/+\RankTg+, Opg+/-\RankTg-, Opg+/-\RankTg+, Opg-/-\RankTg- and Opg-/-\RankTg+ mice) following a protocol (4 injections from post-natal day 1 to 7 at the dose of 50 µg/kg) that mimics those used in onco-pediatric patients. At the end of pediatric growth (1 and half months) and at an adult age (10 months), the bone morphometric and mineral parameters were measured using µCT in the tibia and skull for the different mice. A histologic analysis of the dental and periodontal tissues was also performed. At the end of pediatric growth, a delay in long bone and skull bone growth, a blockage of tooth eruption, some molar root alterations and a neoplasia-like structure associated with incisor development were found. Interestingly, the magnitude of these side effects was reduced by Opg deficiency (Opg-/-) but increased by Rank overexpression (RankTg). Analysis of the skeletal phenotype at ten months confirmed respectively the beneficial and harmful effects of Opg deficiency and Rank overexpression. These results validated the hypothesis that the RANKL signaling activity level in the bone microenvironment is implicated in the modulation of the response to ZOL. Further studies will be necessary to understand the underlying molecular mechanisms, which will help decipher the variability in the effects of N-BPs reported in the human population. SIGNIFICANT STATEMENTS: The present study establishes that in mice the RANKL signaling activity level is a major modulator of the effects and side-effects of bisphosphonates on the individual skeleton during growth. However, the modulatory actions are dependent on the ways in which this level of activity is increased. A decrease in OPG expression is beneficial to the skeletal phenotype observed at the end of growth, while RANK overexpression deteriorates it. Far removed from pediatric treatment, in adults, the skeletal phenotypes initially observed at the end of growth for the different levels of RANKL signaling activity were maintained, although significant improvement was associated only with reductions in OPG expression.

Improving sensitivity of amyloid detection by Congo red stain by using polarizing microscope and avoiding pitfalls.

Systemic amyloidosis is a devastating group of disorders for which there is no current cure. The treatment goal is to reduce the burden of amyloidogenic protein precursors. The treatment is only effective if applied early in the disease process before significant and irreversible end organ damage has taken place. Congo red is still the standard stain used in most histopathology laboratories to identify amyloid material in tissues. The identification of Congophilic amyloid material is challenging because of multiple interfering factors. Here we describe improved sensitivity of identifying Congophilic materials in histologic sections using a metallurgical polarized microscope specifically constructed for polarized microscopy. The microscope is equipped with strain-free optics, matching polarizers, dis-integrated compensators, and a circular mobile stage. Compared to a standard clinical microscope, this setup significantly improves sensitivity of identifying amyloid material in Congo red-stained slides. We also describe the deleterious effect of plastic coverslip which can interfere with the ability to examine the slides under polarized light. We present a series of 10 different patients who had cardiac, brain, and salivary gland biopsies that were either equivocal or deemed negative using a standard clinical microscope but were positive using the equipment described above. These samples were confirmed to be positive by other methods including electron microscopy. We conclude that use of the correct equipment is needed before ruling out amyloidosis in tissue sections.

Lymph Node Mesenchymal and Endothelial Stromal Cells Cooperate via the RANK-RANKL Cytokine Axis to Shape the Sinusoidal Macrophage Niche.

Tissue-resident macrophages are receptive to specific signals concentrated in cellular niches that direct their cell differentiation and maintenance genetic programs. Here, we found that deficiency of the cytokine RANKL in lymphoid tissue organizers and marginal reticular stromal cells of lymph nodes resulted in the loss of the CD169+ sinusoidal macrophages (SMs) comprising the subcapsular and the medullary subtypes. Subcapsular SM differentiation was impaired in mice with targeted RANK deficiency in SMs. Temporally controlled RANK removal in lymphatic endothelial cells (LECs) revealed that lymphatic RANK activation during embryogenesis and shortly after birth was required for the differentiation of both SM subtypes. Moreover, RANK expression by LECs was necessary for SM restoration after inflammation-induced cell loss. Thus, cooperation between mesenchymal cells and LECs shapes a niche environment that supports SM differentiation and reconstitution after inflammation.

sp2-Iminosugar glycolipids as inhibitors of lipopolysaccharide-mediated human dendritic cell activation in vitro and of acute inflammation in mice in vivo.

Glycolipid mimetics consisting of a bicyclic polyhydroxypiperidine-cyclic carbamate core and a pseudoanomeric hydrophobic tail, termed sp2-iminosugar glycolipids (sp2-IGLs), target microglia during neuroinflammatory processes. Here we have synthesized and investigated new variants of sp2-IGLs for their ability to suppress the activation of human monocyte-derived dendritic cells (DCs) by lipopolysaccharide (LPS) signaling through Toll-like receptor 4. We report that the best lead was (1R)-1-dodecylsulfonyl-5N,6O-oxomethylidenenojirimycin (DSO2-ONJ), able to inhibit LPS-induced TNFα production and maturation of DCs. Immunovisualization experiments, using a mannoside glycolipid conjugate (MGC) that also suppress LPS-mediated DC activation as control, evidenced a distinct mode of action for the sp2-IGLs: unlike MGCs, DSO2-ONJ did not elicit internalization of the LPS co-receptor CD14 or induce its co-localization with the Toll-like receptor 4. In a mouse model of LPS-induced acute inflammation, DSO2-ONJ demonstrated anti-inflammatory activity by inhibiting the production of the pro-inflammatory interleukin-6. The ensemble of the data highlights sp2-IGLs as a promising new class of molecules against inflammation by interfering in Toll-like receptor intracellular signaling.