High glucose disrupts oligosaccharide recognition function via competitive inhibition : a potential mechanism for immune dysregulation in diabetes mellitus

Diabetic complications include infection and cardiovascular disease. Within the immune system, host-pathogen and regulatory host-host interactions operate through binding of oligosaccharides by C-type lectin. A number of C-type lectins recognise oligosaccharides rich in mannose and fucose – sugars with similar structures to glucose. This raises the possibility that high glucose conditions in diabetes affect protein-oligosaccharide interactions via competitive inhibition. Mannose binding lectin, soluble DC-SIGN & DC-SIGNR, and surfactant protein D, were tested for carbohydrate binding in the presence of glucose concentrations typical of diabetes, via surface plasmon resonance and affinity chromatography. Complement activation assays were performed in high glucose. DC-SIGN and DC-SIGNR expression in adipose tissues was examined via immunohistochemistry. High glucose inhibited C-type lectin binding to high-mannose glycoprotein and binding of DC-SIGN to fucosylated ligand (blood group B) was abrogated in high glucose. Complement activation via the lectin pathway was inhibited in high glucose and also in high trehalose - a nonreducing sugar with glucoside stereochemistry. DC-SIGN staining was seen on cells with DC morphology within omental and subcutaneous adipose tissues. We conclude that high glucose disrupts C-type lectin function, potentially illuminating new perspectives on susceptibility to infectious and inflammatory disease in diabetes. Mechanisms involve competitive inhibition of carbohydrate-binding within sets of defined proteins, in contrast to broadly indiscriminate, irreversible glycation of proteins

X-Ray Structure of the Human Calreticulin Globular Domain Reveals a Peptide-Binding Area and Suggests a Multi-Molecular Mechanism

In the endoplasmic reticulum, calreticulin acts as a chaperone and a Ca2+-signalling protein. At the cell surface, it mediates numerous important biological effects. The crystal structure of the human calreticulin globular domain was solved at 1.55 Å resolution. Interactions of the flexible N-terminal extension with the edge of the lectin site are consistently observed, revealing a hitherto unidentified peptide-binding site. A calreticulin molecular zipper, observed in all crystal lattices, could further extend this site by creating a binding cavity lined by hydrophobic residues. These data thus provide a first structural insight into the lectin-independent binding properties of calreticulin and suggest new working hypotheses, including that of a multi-molecular mechanism

Wiskott-Aldrich syndrome protein deficiency in innate immune cells leads to mucosal immune dysregulation and colitis in mice

BACKGROUND & AIMS: Immunodeficiency and autoimmune sequelae, including colitis, develop in patients and mice deficient in Wiskott-Aldrich Syndrome protein (WASP), a hematopoietic-specific intracellular signaling molecule that regulates the actin cytoskeleton. Development of colitis in WASP-deficient mice requires lymphocytes; transfer of T cells is sufficient to induce colitis in immunodeficient mice. We investigated the interactions between innate and adaptive immune cells in mucosal regulation during development of T-cell-mediated colitis in mice with WASP-deficient cells of the innate immune system. METHODS: Naïve and/or regulatory CD4(+) T cells were transferred from 129 SvEv mice into RAG-2 deficient (RAG-2 KO) mice or mice lacking WASP and RAG-2 (WRDKO). Animals were observed for the development of colitis; effector and regulatory functions of innate immune and T cells were analyzed with in vivo and in vitro assays. RESULTS: Transfer of unfractionated CD4(+) T cells induced severe colitis in WRDKO, but not RAG-2 KO, mice. Naïve wild-type T cells had higher levels of effector activity and regulatory T cells had reduced suppressive function when transferred into WRDKO mice compared to RAG-2 KO mice. Regulatory T-cell proliferation, generation, and maintenance of FoxP3 expression were reduced in WRDKO recipients, and associated with reduced numbers of CD103(+) tolerogenic dendritic cells and levels of interleukin (IL)-10. Administration of IL-10 prevented induction of colitis following transfer of T cells into WRDKO mice. CONCLUSIONS: Defective interactions between WASP-deficient innate immune cells and normal T cells disrupt mucosal regulation, potentially by altering the functions of tolerogenic dendritic cells, production of IL-10, and homeostasis of regulatory T cells

Fundamental role of C1q in autoimmunity and inflammation

C1q, historically viewed as the initiating component of the classical complement pathway, also exhibits a variety of complement-independent activities in both innate and acquired immunity. Recent studies focusing on C1q\u27s suppressive role in the immune system have provided new insight into how abnormal C1q expression and bioactivity may contribute to autoimmunity. In particular, molecular networks involving C1q interactions with cell surface receptors and other ligands are emerging as mechanisms involved in C1q\u27s modulation of immunity. Here, we discuss the role of C1q in controlling immune cell function, including recently elucidated mechanisms of action, and suggest how these processes are critical for maintaining tissue homeostasis under steady-state conditions and in preventing autoimmunity

670-nm light treatment reduces complement propagation following retinal degeneration

AIM: Complement activation is associated with the pathogenesis of age-related macular degeneration (AMD). We aimed to investigate whether 670-nm light treatment reduces the propagation of complement in a light-induced model of atrophic AMD. METHODS: Sprague–Dawley (SD) rats were pretreated with 9 J/cm(2) 670-nm light for 3 minutes daily over 5 days; other animals were sham treated. Animals were exposed to white light (1,000 lux) for 24 h, after which animals were kept in dim light (5 lux) for 7 days. Expression of complement genes was assessed by quantitative polymerase chain reaction (qPCR), and immunohistochemistry. Counts were made of C3-expressing monocytes/microglia using in situ hybridization. Photoreceptor death was also assessed using outer nuclear layer (ONL) thickness measurements, and oxidative stress using immunohistochemistry for 4-hydroxynonenal (4-HNE). RESULTS: Following light damage, retinas pretreated with 670-nm light had reduced immunoreactivity for the oxidative damage maker 4-HNE in the ONL and outer segments, compared to controls. In conjunction, there was significant reduction in retinal expression of complement genes C1s, C2, C3, C4b, C3aR1, and C5r1 following 670 nm treatment. In situ hybridization, coupled with immunoreactivity for the marker ionized calcium binding adaptor molecule 1 (IBA1), revealed that C3 is expressed by infiltrating microglia/monocytes in subretinal space following light damage, which were significantly reduced in number after 670 nm treatment. Additionally, immunohistochemistry for C3 revealed a decrease in C3 deposition in the ONL following 670 nm treatment. CONCLUSIONS: Our data indicate that 670-nm light pretreatment reduces lipid peroxidation and complement propagation in the degenerating retina. These findings have relevance to the cellular events of complement activation underling the pathogenesis of AMD, and highlight the potential of 670-nm light as a non-invasive anti-inflammatory therapy

Synthesis and propagation of complement C3 by microglia/monocytes in the aging retina

INTRODUCTION Complement activation is thought to contribute to the pathogenesis of age-related macular degeneration (AMD), which may be mediated in part by para-inflammatory processes. We aimed to investigate the expression and localization of C3, a crucial component of the complement system, in the retina during the course of aging. METHODS SD rats were born and reared in low-light conditions, and euthanized at post-natal (P) days 100, 450, or 750. Expression of C3, IBA1, and Ccl- and Cxcl- chemokines was assessed by qPCR, and in situ hybridization. Thickness of the ONL was assessed in retinal sections as a measure of photoreceptor loss, and counts were made of C3-expressing monocytes. RESULTS C3 expression increased significantly at P750, and correlated with thinning of the ONL, at P750, and up-regulation of GFAP. In situ hybridization showed that C3 was expressed by microglia/monocytes, mainly from within the retinal vasculature, and occasionally the ONL. The number of C3-expressing microglia increased significantly by P750, and coincided spatiotemporally with thinning of the ONL, and up-regulation of Ccl- and Cxcl- chemokines. CONCLUSIONS Our data suggest that recruited microglia/monocytes contribute to activation of complement in the aging retina, through local expression of C3 mRNA. C3 expression coincides with age-related thinning of the ONL at P750, although it is unclear whether the C3-expressing monocytes are a cause or consequence. These findings provide evidence of activation of complement during natural aging, and may have relevance to cellular events underling the pathogenesis of age-related retinal diseases.Funding provided by Australian Research Council Centres of Excellence Program Grant (CE0561903)

Investigation of Complement Activation Product C4d as a Diagnostic and Prognostic Biomarker for Lung Cancer

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3776260/[EN] Background There is a medical need for diagnostic biomarkers in lung cancer. We evaluated the diagnostic performance of complement activation fragments. Methods We assessed complement activation in four bronchial epithelial and seven lung cancer cell lines. C4d, a degradation product of complement activation, was determined in 90 primary lung tumors; bronchoalveolar lavage supernatants from patients with lung cancer (n = 50) and nonmalignant respiratory diseases (n = 22); and plasma samples from advanced (n = 50) and early lung cancer patients (n = 84) subjects with inflammatory lung diseases (n = 133), and asymptomatic individuals enrolled in a lung cancer computed tomography screening program (n = 190). Two-sided P values were calculated by Mann-Whitney U test. Results Lung cancer cells activated the classical complement pathway mediated by C1q binding that was inhibited by phosphomonoesters. Survival was decreased in patients with high C4d deposition in tumors (hazard ratio [HR] = 3.06; 95% confidence interval [CI] = 1.18 to 7.91). C4d levels were increased in bronchoalveolar lavage fluid from lung cancer patients compared with patients with nonmalignant respiratory diseases (0.61 +/- 0.87 vs 0.16 +/- 0.11 mu g/mL; P < .001). C4d levels in plasma samples from lung cancer patients at both advanced and early stages were also increased compared with control subjects (4.13 +/- 2.02 vs 1.86 +/- 0.95 mu g/mL, P < 0.001; 3.18 +/- 3.20 vs 1.13 +/- 0.69 mu g/mL, P < .001, respectively). C4d plasma levels were associated with shorter survival in patients at advanced (HR = 1.59; 95% CI = 0.97 to 2.60) and early stages (HR = 5.57; 95% CI = 1.60 to 19.39). Plasma C4d levels were reduced after surgical removal of lung tumors (P < .001) and were associated with increased lung cancer risk in asymptomatic individuals with (n = 32) or without lung cancer (n = 158) (odds ratio = 4.38; 95% CI = 1.61 to 11.93). Conclusions Complement fragment C4d may serve as a biomarker for early diagnosis and prognosis of lung cancer.This work was supported by UTE project CIMA; the Spanish Government (grant numbers ISCIII-RTICC RD06/0020/0066, RD06/0020/1024, RD12/0036/0025, RD12/0036/0040, RD12/0036/0062, PI08/0923, PI10/01652, PI10/00166, and PI11/00618); the European Regional Development Fund; the European Community’s Seventh Framework Programme (HEALTH-F2-2010-258677- CURELUNG); and the Early Detection Research Network from the National Cancer Institute (grant number U01 CA152662). This work was supported (in part) by a grant (RD12/0036/XXXX) from Red Temática de Investigación Cooperativa en Cáncer, Instituto de Salud Carlos III, Spanish Ministry of Economy and Competitiveness & European Regional Development Fund “Una manera de hacer Europa”.Jantus Lewintre, E. (2013). Investigation of Complement Activation Product C4d as a Diagnostic and Prognostic Biomarker for Lung Cancer. JNCI: Journal of the National Cancer Institute. 105:1385-1393. https://doi.org/10.1093/jnci/djt205S1385139310

Open Source ImmGen: network perspective on metabolic diversity among mononuclear phagocytes

We dissect metabolic variability of mononuclear phagocyte (MNP) subpopulations across different tissues through integrative analysis of three large scale datasets. Specifically, we introduce ImmGen MNP Open Source dataset that profiled 337 samples and extended previous ImmGen effort which included 202 samples of mononuclear phagocytes and their progenitors. Next, we analysed Tabula Muris Senis dataset to extract data for 51,364 myeloid cells from 18 tissues. Taken together, a compendium of data assembled in this work covers phagocytic populations found across 38 different tissues. To analyse common metabolic features, we developed novel network-based computational approach for unbiased identification of key metabolic subnetworks based on cellular transcriptional profiles in large-scale datasets. Using ImmGen MNP Open Source dataset as baseline, we define 9 metabolic subnetworks that encapsulate the metabolic differences within mononuclear phagocytes, and demonstrate that these features are robustly found across all three datasets, including lipid metabolism, cholesterol biosynthesis, glycolysis, and a set of fatty acid related metabolic pathways, as well as nucleotide and folate metabolism. We systematically define major features specific to macrophage and dendritic cell subpopulations. Among other things, we find that cholesterol synthesis appears particularly active within the migratory dendritic cells. We demonstrate that interference with this pathway through statins administration diminishes migratory capacity of the dendritic cells in vivo. This result demonstrates the power of our approach and highlights importance of metabolic diversity among mononuclear phagocytes

Network analysis of large-scale ImmGen and Tabula Muris datasets highlights metabolic diversity of tissue mononuclear phagocytes

The diversity of mononuclear phagocyte (MNP) subpopulations across tissues is one of the key physiological characteristics of the immune system. Here, we focus on understanding the metabolic variability of MNPs through metabolic network analysis applied to three large-scale transcriptional datasets: we introduce (1) an ImmGen MNP open-source dataset of 337 samples across 26 tissues; (2) a myeloid subset of ImmGen Phase I dataset (202 MNP samples); and (3) a myeloid mouse single-cell RNA sequencing (scRNA-seq) dataset (51,364 cells) assembled based on Tabula Muris Senis. To analyze such large-scale datasets, we develop a network-based computational approach, genes and metabolites (GAM) clustering, for unbiased identification of the key metabolic subnetworks based on transcriptional profiles. We define 9 metabolic subnetworks that encapsulate the metabolic differences within MNP from 38 different tissues. Obtained modules reveal that cholesterol synthesis appears particularly active within the migratory dendritic cells, while glutathione synthesis is essential for cysteinyl leukotriene production by peritoneal and lung macrophages