Complement 3 signaling is necessary for the developmental refinement of olfactory bulb circuitry

AbstractThe olfactory system depends upon organizational maps that are developmentally refined and maintained, however the cellular and molecular mechanisms that underlie these processes are unknown. Studies have shown that microglia and complement molecules are important for the developmental refinement of circuitry within the visual system, thus we asked whether they played a similar role in the olfactory system through the formation of the olfactory bulb (OB) maps, the glomerular and intrabulbar maps. Our findings revealed that microglia in mature animals engulf olfactory sensory neuron (OSN) axons and the synaptic terminals of tufted cells in the glomerular and intrabulbar maps respectively, suggesting microglia could anatomically shape the mature OB circuitry. To determine the mechanisms underlying this axonal pruning activity we used complement 3 (C3) and complement receptor 3 (CR3) knockout mice to investigate if C3 signaling was necessary for precise OB map development. Our results demonstrate that glomerular and intrabulbar map disorganization as typically present in early postnatal mice persists into adulthood when C3 signaling is disrupted. These data clearly establish the C3/CR3 pathway as necessary for the proper developmental refinement of both olfactory maps. We further present the olfactory system as a unique platform to study the role of glia in the development and adult refinement of regenerating circuits.

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Utilization of CD11b Knockout Mice to Characterize the Role of Complement Receptor 3 (CR3, CD11b/CD18) in the Growth of Mycobacterium tuberculosis in Macrophages

Cellular Immunology ◽

10.1006/cimm.2000.1710 ◽

2000 ◽

Vol 205 (1) ◽

pp. 13-23 ◽

Cited By ~ 40

Author(s):

Maico D. Melo ◽

Ian R. Catchpole ◽

Graham Haggar ◽

Richard W. Stokes

Keyword(s):

Mycobacterium Tuberculosis ◽

Knockout Mice ◽

Complement Receptor ◽

Complement Receptor 3

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β-Glucan enhances complement-mediated hematopoietic recovery after bone marrow injury

Blood ◽

10.1182/blood-2005-07-2705 ◽

2006 ◽

Vol 107 (2) ◽

pp. 835-840 ◽

Cited By ~ 36

Author(s):

Daniel E. Cramer ◽

Daniel J. Allendorf ◽

Jarek T. Baran ◽

Richard Hansen ◽

Jose Marroquin ◽

...

Keyword(s):

Bone Marrow ◽

Mouse Serum ◽

Complement Receptor ◽

Dependent Manner ◽

Complement Receptor 3 ◽

Total Body ◽

Stimulating Factor ◽

Sublethal Irradiation ◽

Classic Pathway

AbstractMyelotoxic injury in the bone marrow (BM) as a consequence of total body irradiation (TBI) or granulocyte colony-stimulating factor (G-CSF) mobilization results in the deposition of iC3b on BM stroma (stroma-iC3b). In the present study, we have examined how stroma-iC3b interacts with hematopoietic progenitor cells (HPCs) and the role of complement (C) and complement receptor 3 (CR3) in BM injury/repair. We demonstrate here that stroma-iC3b tethers HPCs via the inserted (I) domain of HPC complement receptor 3 (CR3, CD11b/CD18, Mac-1). Following irradiation, stroma-iC3b was observed in the presence of purified IgM and normal mouse serum (NMS), but not serum from Rag-2-/- mice, implicating a role for antibody (Ab) and the classic pathway of C activation. Furthermore, a novel role for soluble yeast β-glucan, a ligand for the CR3 lectin-like domain (LLD), in the priming of CR3+ HPC is suggested. Soluble yeast β-glucan could enhance the proliferation of tethered HPCs, promote leukocyte recovery following sublethal irradiation, and increase the survival of lethally irradiated animals following allogeneic HPC transplantation in a CR3-dependent manner. Taken together, these observations suggest a novel role for C, CR3, and β-glucan in the restoration of hematopoiesis following injury. (Blood. 2006;107:835-840)

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Role of complement and Fcγ receptors in the protective activity of the long pentraxin PTX3 against Aspergillus fumigatus

Blood ◽

10.1182/blood-2009-12-258376 ◽

2010 ◽

Vol 116 (24) ◽

pp. 5170-5180 ◽

Cited By ~ 133

Author(s):

Federica Moalli ◽

Andrea Doni ◽

Livija Deban ◽

Teresa Zelante ◽

Silvia Zagarella ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Alternative Pathway ◽

Complement Receptor ◽

Protective Activity ◽

Opsonic Activity ◽

Reactive Protein ◽

Complement Receptor 3 ◽

Dependent Pathway

AbstractPentraxin 3 (PTX3) is a soluble pattern recognition molecule playing a nonredundant role in resistance against Aspergillus fumigatus. The present study was designed to investigate the molecular pathways involved in the opsonic activity of PTX3. The PTX3 N-terminal domain was responsible for conidia recognition, but the full-length molecule was necessary for opsonic activity. The PTX3-dependent pathway of enhanced neutrophil phagocytic activity involved complement activation via the alternative pathway; Fcγ receptor (FcγR) IIA/CD32 recognition of PTX3-sensitized conidia and complement receptor 3 (CR3) activation; and CR3 and CD32 localization to the phagocytic cup. Gene targeted mice (ptx3, FcR common γ chain, C3, C1q) validated the in vivo relevance of the pathway. In particular, the protective activity of exogenous PTX3 against A fumigatus was abolished in FcR common γ chain-deficient mice. Thus, the opsonic and antifungal activity of PTX3 is at the crossroad between complement, complement receptor 3-, and FcγR-mediated recognition. Because short pentraxins (eg, C-reactive protein) interact with complement and FcγR, the present results may have general significance for the mode of action of these components of the humoral arm of innate immunity.

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CD44-mediated phagocytosis induces inside-out activation of complement receptor-3 in murine macrophages

Blood ◽

10.1182/blood-2007-02-076539 ◽

2007 ◽

Vol 110 (13) ◽

pp. 4492-4502 ◽

Cited By ~ 39

Author(s):

Eric Vachon ◽

Raiza Martin ◽

Vivian Kwok ◽

Vera Cherepanov ◽

Chung-Wai Chow ◽

...

Keyword(s):

Intracellular Signaling ◽

Immunofluorescence Microscopy ◽

Complement Receptor ◽

Fcγ Receptors ◽

Intracellular Signaling Pathways ◽

Large Particles ◽

Complement Receptor 3 ◽

Inside Out ◽

Β2 Integrins

Diverse receptors, including Fcγ receptors and β2 integrins (complement receptor-3 [CR3], CD11b/CD18), have been implicated in phagocytosis, but their distinct roles and interactions with other receptors in particle engulfment are not well defined. CD44, a transmembrane adhesion molecule involved in binding and metabolism of hyaluronan, may have additional functions in regulation of inflammation and phagocytosis. We have recently reported that CD44 is a fully competent phagocytic receptor that is able to trigger ingestion of large particles by macrophages. Here, we investigated the role of coreceptors and intracellular signaling pathways in modulation of CD44-mediated phagocytosis. Using biotinylated erythrocytes coated with specific antibodies (anti-CD44–coated erythrocytes [Ebabs]) as the phagocytic prey, we determined that CD44-mediated phagocytosis is reduced by 45% by a blocking CD11b antibody. Further, CD44-mediated phagocytosis was substantially (42%) reduced in CD18-null mice. Immunofluorescence microscopy revealed that CD11b is recruited to the phagocytic cup. The mechanism of integrin activation and mobilization involved activation of the GTPase Rap1. CD44-mediated phagocytosis was also sensitive to the extracellular concentration of the divalent cation Mg2+ but not Ca2+. In addition, buffering of intracellular Ca2+ did not affect CD44-mediated phagocytosis. Taken together, these data suggest that CD44 stimulation induces inside-out activation of CR3 through the GTPase Rap1.

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Dectin-1 Is A Major β-Glucan Receptor On Macrophages

Journal of Experimental Medicine ◽

10.1084/jem.20020470 ◽

2002 ◽

Vol 196 (3) ◽

pp. 407-412 ◽

Cited By ~ 656

Author(s):

Gordon D. Brown ◽

Philip R. Taylor ◽

Delyth M. Reid ◽

Janet A. Willment ◽

David L. Williams ◽

...

Keyword(s):

Monoclonal Antibody ◽

Drug Design ◽

Mannose Receptor ◽

Therapeutic Drug ◽

Complement Receptor ◽

Macrophage Mannose Receptor ◽

Immunomodulatory Properties ◽

Complement Receptor 3

Zymosan is a β-glucan– and mannan-rich particle that is widely used as a cellular activator for examining the numerous responses effected by phagocytes. The macrophage mannose receptor (MR) and complement receptor 3 (CR3) have historically been considered the major macrophage lectins involved in the nonopsonic recognition of these yeast-derived particles. Using specific carbohydrate inhibitors, we show that a β-glucan receptor, but not the MR, is a predominant receptor involved in this process. Furthermore, nonopsonic zymosan binding was unaffected by genetic CD11b deficiency or a blocking monoclonal antibody (mAb) against CR3, demonstrating that CR3 was not the β-glucan receptor mediating this activity. To address the role of the recently described β-glucan receptor, Dectin-1, we generated a novel anti–Dectin-1 mAb, 2A11. Using this mAb, we show here that Dectin-1 was almost exclusively responsible for the β-glucan–dependent, nonopsonic recognition of zymosan by primary macro-phages. These findings define Dectin-1 as the leukocyte β-glucan receptor, first described over 50 years ago, and resolves the long-standing controversy regarding the identity of this important molecule. Furthermore, these results identify Dectin-1 as a new target for examining the immunomodulatory properties of β-glucans for therapeutic drug design.

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Role of intraglomerular circuits in shaping temporally structured responses to naturalistic inhalation-driven sensory input to the olfactory bulb

Journal of Neurophysiology ◽

10.1152/jn.00394.2014 ◽

2015 ◽

Vol 113 (9) ◽

pp. 3112-3129 ◽

Cited By ~ 11

Author(s):

Ryan M. Carey ◽

William Erik Sherwood ◽

Michael T. Shipley ◽

Alla Borisyuk ◽

Matt Wachowiak

Keyword(s):

Olfactory Bulb ◽

Sensory Neuron ◽

Temporal Structure ◽

Decay Kinetics ◽

Response Dynamics ◽

Output Neurons ◽

Tufted Cells ◽

Slow Onset ◽

Feedforward Inhibition

Olfaction in mammals is a dynamic process driven by the inhalation of air through the nasal cavity. Inhalation determines the temporal structure of sensory neuron responses and shapes the neural dynamics underlying central olfactory processing. Inhalation-linked bursts of activity among olfactory bulb (OB) output neurons [mitral/tufted cells (MCs)] are temporally transformed relative to those of sensory neurons. We investigated how OB circuits shape inhalation-driven dynamics in MCs using a modeling approach that was highly constrained by experimental results. First, we constructed models of canonical OB circuits that included mono- and disynaptic feedforward excitation, recurrent inhibition and feedforward inhibition of the MC. We then used experimental data to drive inputs to the models and to tune parameters; inputs were derived from sensory neuron responses during natural odorant sampling (sniffing) in awake rats, and model output was compared with recordings of MC responses to odorants sampled with the same sniff waveforms. This approach allowed us to identify OB circuit features underlying the temporal transformation of sensory inputs into inhalation-linked patterns of MC spike output. We found that realistic input-output transformations can be achieved independently by multiple circuits, including feedforward inhibition with slow onset and decay kinetics and parallel feedforward MC excitation mediated by external tufted cells. We also found that recurrent and feedforward inhibition had differential impacts on MC firing rates and on inhalation-linked response dynamics. These results highlight the importance of investigating neural circuits in a naturalistic context and provide a framework for further explorations of signal processing by OB networks.

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Molecular mechanism of leukocidin GH–integrin CD11b/CD18 recognition and species specificity

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1913690116 ◽

2019 ◽

Vol 117 (1) ◽

pp. 317-327 ◽

Cited By ~ 1

Author(s):

Nikolina Trstenjak ◽

Dalibor Milić ◽

Melissa A. Graewert ◽

Harald Rouha ◽

Dmitri Svergun ◽

...

Keyword(s):

Animal Models ◽

Molecular Mechanism ◽

Molecular Mechanisms ◽

Cytolytic Activity ◽

Receptor Interaction ◽

Complement Receptor ◽

Microbial Pathogenesis ◽

Complement Receptor 3 ◽

Binding Interfaces ◽

Insight Into

Host–pathogen interactions are central to understanding microbial pathogenesis. The staphylococcal pore-forming cytotoxins hijack important immune molecules but little is known about the underlying molecular mechanisms of cytotoxin–receptor interaction and host specificity. Here we report the structures of a staphylococcal pore-forming cytotoxin, leukocidin GH (LukGH), in complex with its receptor (the α-I domain of complement receptor 3, CD11b-I), both for the human and murine homologs. We observe 2 binding interfaces, on the LukG and the LukH protomers, and show that human CD11b-I induces LukGH oligomerization in solution. LukGH binds murine CD11b-I weakly and is inactive toward murine neutrophils. Using a LukGH variant engineered to bind mouse CD11b-I, we demonstrate that cytolytic activity does not only require binding but also receptor-dependent oligomerization. Our studies provide an unprecedented insight into bicomponent leukocidin–host receptor interaction, enabling the development of antitoxin approaches and improved animal models to explore these approaches.

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Abstract 5290: Cypher Long but not Short Isoform Specific Knockout Mice Result in Cardiac Signaling Defects and Dilated Cardiomyopathy

Circulation ◽

10.1161/circ.118.suppl_18.s_521 ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Hongqiang Cheng ◽

Ming Zheng ◽

Farah Sheikh ◽

Kunfu Ouyang ◽

Li Cui ◽

...

Keyword(s):

Dilated Cardiomyopathy ◽

Knockout Mice ◽

Molecular Mechanisms ◽

Heart Function ◽

Gene Mutations ◽

Splice Isoforms ◽

Functional Roles ◽

Deficient Mice

Our previous studies have demonstrated that Cypher, a PDZ-LIM protein localized at the Z line, plays a pivotal role in heart function. We recently identified long and short splice isoforms of Cypher, which are characterized by the presence and absence of LIM domains, respectively. The LIM domain of Cypher is thought to be involved in signaling, based on its ability to directly interact with signaling proteins. In human patients with dilated cardiomyopathy (DCM) we discovered Cypher gene mutations, which affect either long or short isoform or both isoforms. However, the precise molecular mechanisms underlying the role of Cypher isoforms in DCM remain unclear. To determine the role of Cypher isoforms in cardiac signaling and disease in vivo , we generated two Cypher isoform specific knockout mice. Selective ablation of Cypher long isoforms in mice resulted in partial neonatal lethality. However, hearts from viable Cypher long isoform deficient mice displayed Z line abnormalities and decreased cardiomyocyte widths, which resulted in a progressive form of DCM, characterized by fibrosis, calcification and lethality. The effects on cardiac function and disease observed in long-isoform specific Cypher knockout mice were preceded by significant decreases in cardiac protein kinase C and extracellular signal-regulated kinase signaling. These results are in contrast to Cypher short isoform deficient mice, which were viable with no overt cardiac morphology and signaling abnormalities. These results reveal distinct functional roles for Cypher isoforms in the heart as well as shed light into the molecular mechanisms underlying dilated cardiomyopathy.

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GATA-2 Regulates Dendritic Cell Differentiation

Blood ◽

10.1182/blood.v126.23.2382.2382 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2382-2382

Author(s):

Koichi Onodera ◽

Tohru Fujiwara ◽

Yasushi Onishi ◽

Ari Itoh-Nakadai ◽

Yoko Okitsu ◽

...

Keyword(s):

Dendritic Cell ◽

Cell Line ◽

Knockout Mice ◽

Research Funding ◽

Molecular Mechanisms ◽

Conditional Knockout ◽

Luciferase Reporter ◽

Chip Analysis

Abstract (Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms. (Method) Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology). (Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP. To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells. (Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. Disclosures Fujiwara: Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.

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