Publications by authors named "Trent M Woodruff"

151 Publications

Glucose clearance and uptake is increased in the SOD1 mouse model of amyotrophic lateral sclerosis through an insulin-independent mechanism.

FASEB J 2021 07;35(7):e21707

School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, QLD, Australia.

Metabolic disturbances are associated with the progression of the neurodegenerative disorder, amyotrophic lateral sclerosis (ALS). However, the molecular events that drive energy imbalances in ALS are not completely understood. In this study, we aimed to elucidate deficits in energy homeostasis in the SOD1 mouse model of ALS. SOD1 mice and their wild-type littermates underwent indirect calorimetry and intraperitoneal glucose/insulin tolerance tests at both the onset and mid-symptomatic stages of the disease. Glucose uptake and the plasma glucoregulatory hormone profiles were analyzed. Pancreatic islet cell mass and function were assessed by measuring hormone concentrations and secretion in isolated islets, and pancreatic α- and β-cell immunoreactive areas. Finally, we profiled liver glycogen metabolism by measuring glucagon concentrations and liver metabolic gene expressions. We identified that mid-symptomatic SOD1 mice have increased oxygen consumption and faster exogenous glucose uptake, despite presenting with normal insulin tolerance. The capacity for pancreatic islets to secrete insulin appears intact, however, islet cell insulin concentrations and β-cell mass were reduced. Fasting glucose homeostasis was also disturbed, along with increased liver glycogen stores, despite elevated circulating glucagon, suggesting that glucagon signaling is impaired. Metabolic gene expression profiling of livers indicated that glucose cannot be utilized efficiently in SOD1 mice. Overall, we demonstrate that glucose homeostasis and uptake are altered in SOD1 mice, which is linked to an increase in insulin-independent glucose uptake, and a loss of β-cells, insulin production, and glucagon sensitivity. This suggests that the hormonal regulation of glucose concentrations may contribute to the progression of disease in this ALS mouse model.
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http://dx.doi.org/10.1096/fj.202002450RDOI Listing
July 2021

C5a-C5aR1 Axis Activation Drives Envenomation Immunopathology by the Snake .

Front Immunol 2021 15;12:652242. Epub 2021 Apr 15.

Immunochemistry Laboratory, Instituto Butantan, São Paulo, Brazil.

Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement's contribution to the physiopathogenesis of envenomation. We found that venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by and possibly by other snake venoms.
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http://dx.doi.org/10.3389/fimmu.2021.652242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8082402PMC
April 2021

Processed foods drive intestinal barrier permeability and microvascular diseases.

Sci Adv 2021 Mar 31;7(14). Epub 2021 Mar 31.

Department of Diabetes, Central Clinical School, Alfred Medical Research and Education Precinct, Monash University, Melbourne, Victoria, Australia.

Intake of processed foods has increased markedly over the past decades, coinciding with increased microvascular diseases such as chronic kidney disease (CKD) and diabetes. Here, we show in rodent models that long-term consumption of a processed diet drives intestinal barrier permeability and an increased risk of CKD. Inhibition of the advanced glycation pathway, which generates Maillard reaction products within foods upon thermal processing, reversed kidney injury. Consequently, a processed diet leads to innate immune complement activation and local kidney inflammation and injury via the potent proinflammatory effector molecule complement 5a (C5a). In a mouse model of diabetes, a high resistant starch fiber diet maintained gut barrier integrity and decreased severity of kidney injury via suppression of complement. These results demonstrate mechanisms by which processed foods cause inflammation that leads to chronic disease.
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http://dx.doi.org/10.1126/sciadv.abe4841DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8011970PMC
March 2021

The "C3aR Antagonist" SB290157 is a Partial C5aR2 Agonist.

Front Pharmacol 2020 21;11:591398. Epub 2021 Jan 21.

School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia.

Innate immune complement activation generates the C3 and C5 protein cleavage products C3a and C5a, defined classically as anaphylatoxins. C3a activates C3aR, while C5a activates two receptors (C5aR1 and C5aR2) to exert their immunomodulatory activities. The non-peptide compound, SB290157, was originally reported in 2001 as the first C3aR antagonist. In 2005, the first report on the non-selective nature of SB290157 was published, where the compound exerted clear agonistic, not antagonistic, activity in variety of cells. Other studies also documented the non-selective activities of this drug in vivo. These findings severely hamper data interpretation regarding C3aR when using this compound. Unfortunately, given the dearth of C3aR inhibitors, SB290157 still remains widely used to explore C3aR biology (>70 publications to date). Given these issues, in the present study we aimed to further explore SB290157's pharmacological selectivity by screening the drug against three human anaphylatoxin receptors, C3aR, C5aR1 and C5aR2, using cell models. We identified that SB290157 exerts partial agonist activity at C5aR2 by mediating -arrestin recruitment at higher compound doses. This translated to a functional outcome in both human and mouse primary macrophages, where SB290157 significantly dampened C5a-induced ERK signaling. We also confirmed that SB290157 acts as a potent agonist at human C3aR in transfected cells, but as an antagonist in primary human macrophages. Our results therefore provide even more caution against using SB290157 as a research tool to explore C3aR function. Given the reported immunomodulatory and anti-inflammatory activities of C5aR2 agonism, any function observed with SB290157 could be due to these off-target activities.
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http://dx.doi.org/10.3389/fphar.2020.591398DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7859635PMC
January 2021

An Immunoregulatory Role for Complement Receptors in Murine Models of Breast Cancer.

Antibodies (Basel) 2021 Jan 8;10(1). Epub 2021 Jan 8.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, QLD 4072, Australia.

The complement system has demonstrated roles in regulating tumor growth, although these may differ between tumor types. The current study used two murine breast cancer models (EMT6 and 4T1) to investigate whether pharmacological targeting of receptors for complement proteins C3a (C3aR) and C5a (C5aR1) is protective in murine breast cancer models. In contrast to prior studies in other tumor models, treatment with the selective C5aR1 antagonist PMX53 had no effect on tumor growth. However, treatment of mice with a dual C3aR/C5aR1 agonist (YSFKPMPLaR) significantly slowed mammary tumor development and progression. Examination of receptor expression by quantitative polymerase chain reaction (qPCR) analysis showed very low levels of mRNA expression for either or by EMT6 or 4T1 mammary carcinoma cell lines compared with the J774 macrophage line or bone marrow-derived macrophages. Moreover, flow cytometric analysis found no evidence of C3aR or C5aR1 protein expression by either EMT6 or 4T1 cells, leading us to hypothesize that the tumor inhibitory effects of the dual agonist are indirect, possibly via regulation of the anti-tumor immune response. This hypothesis was supported by flow cytometric analysis of tumor infiltrating leukocyte populations, which demonstrated a significant increase in T lymphocytes in mice treated with the C3aR/C5aR1 agonist. These results support an immunoregulatory role for complement receptors in primary murine mammary carcinoma models. They also suggest that complement activation peptides can influence the anti-tumor response in different ways depending on the cancer type, the host immune response to the tumor and levels of endogenous complement activation within the tumor microenvironment.
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http://dx.doi.org/10.3390/antib10010002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7838807PMC
January 2021

Chemical synthesis and characterisation of the complement C5 inhibitory peptide zilucoplan.

Amino Acids 2021 Jan 4;53(1):143-147. Epub 2021 Jan 4.

School of Biomedical Sciences, The University of Queensland, Brisbane, Australia.

The complement component C5 inhibitory peptide zilucoplan is currently in phase III clinical trials for myasthenia gravis (MG). Despite being at an advanced stage of clinical development, there have been no published reports in the literature detailing its chemical synthesis. In this work, we describe an approach for the chemical synthesis of zilucoplan and validate that the synthesised compound blocks LPS-induced C5a production from human blood.
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http://dx.doi.org/10.1007/s00726-020-02921-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7781173PMC
January 2021

Tissue-Type Plasminogen Activator and Tenecteplase-Mediated Increase in Blood Brain Barrier Permeability Involves Cell Intrinsic Complement.

Front Neurol 2020 8;11:577272. Epub 2020 Dec 8.

Molecular Neurotrauma and Haemostasis, Australian Centre for Blood Diseases, Monash University, Melbourne, VIC, Australia.

Tissue-type plasminogen activator (t-PA) has been the mainstay of therapeutic thrombolysis for patients with acute ischaemic stroke (AIS). However, t-PA can cause devastating intracerebral hemorrhage. t-PA can also influence the CNS in part by modulation of BBB permeability. Complement activation also occurs after AIS and has also been reported to increase BBB permeability. The complement components, C3 and C5, can also be activated by t-PA via plasmin formation and cell intrinsic complement may be involved in this process. Tenecteplase (TNK-tPA) is a t-PA variant with a longer plasma half-life, yet the ability of TNK-tPA to modulate the BBB and complement is less clear. To evaluate the effect of C5 and C5a-receptor 1 (C5aR1) inhibitors on t-PA- and TNK-tPA-mediated opening of the BBB. We used an model of the BBB where human brain endothelial cells and human astrocytes were co-cultured on the opposite sides of a porous membrane assembled in transwell inserts. The luminal (endothelial) compartment was stimulated with t-PA or TNK-tPA together with plasminogen, in the presence of PMX205 (a non-competitive C5aR1 antagonist), Avacopan (a competitive C5aR1 antagonist) or Eculizumab (a humanized monoclonal inhibitor of human C5). BBB permeability was assessed 5 and 24 h later. Immunofluorescence was also used to detect changes in C5 and C5aR1 expression in endothelial cells and astrocytes. PMX205, but not Avacopan or Eculizumab, blocked t-PA-mediated increase in BBB permeability at both the 5 and 24 h time points. PMX205 also blocked TNK-tPA-mediated increase in BBB permeability. Immunofluorescence analysis revealed intracellular staining of C5 in both cell types. C5aR1 expression was also detected on the cell surfaces and also located intracellularly in both cell types. t-PA and TNK-tPA-mediated increase in BBB permeability involves C5aR1 receptor activation from cell-derived C5a. Selective inhibitors of C5aR1 may have therapeutic potential in AIS.
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http://dx.doi.org/10.3389/fneur.2020.577272DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753024PMC
December 2020

TDP-43 Puts the STING in ALS.

Trends Neurosci 2021 02 20;44(2):81-82. Epub 2020 Dec 20.

School of Biomedical Sciences, University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia; Queensland Brain Institute, University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia. Electronic address:

In a recent study, Yu et al. demonstrated that TAR DNA-binding protein of 43 kDa (TDP-43) causes inflammation in amyotrophic lateral sclerosis (ALS) by triggering mitochondrial (mt)DNA release into the cytoplasm, which subsequently activates the cytoplasmic DNA-sensing cyclic GMP-AMP synthase (cGAS)/stimulator of interferon genes (STING) pathway. These results suggest that inhibition of cGAS/STING could help mitigate inflammation-related neuropathology in ALS.
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http://dx.doi.org/10.1016/j.tins.2020.12.001DOI Listing
February 2021

Is the C3a receptor antagonist SB290157 a useful pharmacological tool?

Br J Pharmacol 2020 12 18;177(24):5677-5678. Epub 2020 Oct 18.

School of Biomedical Sciences, The University of Queensland, St Lucia, Queensland, Australia.

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http://dx.doi.org/10.1111/bph.15264DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7707084PMC
December 2020

Monocytes and neutrophils are associated with clinical features in amyotrophic lateral sclerosis.

Brain Commun 2020 14;2(1):fcaa013. Epub 2020 Feb 14.

School of Biomedical Sciences, The University of Queensland, Brisbane, Queensland 4072, Australia.

Immunity has emerged as a key player in neurodegenerative diseases such as amyotrophic lateral sclerosis, with recent studies documenting aberrant immune changes in patients and animal models. A challenging aspect of amyotrophic lateral sclerosis research is the heterogeneous nature of the disease. In this study, we investigate the associations between peripheral blood myeloid cell populations and clinical features characteristic of amyotrophic lateral sclerosis. Peripheral blood leukocytes from 23 healthy controls and 48 patients with amyotrophic lateral sclerosis were analysed to measure myeloid cell alterations. The proportion of monocytes (classical, intermediates and non-classical subpopulations) and neutrophils, as well as the expression of select surface markers, were quantitated using flow cytometry. Given the heterogeneous nature of amyotrophic lateral sclerosis, multivariable linear analyses were performed to investigate associations between patients' myeloid profile and clinical features, such as the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale, bulbar subscore of the Revised Amyotrophic Lateral Sclerosis Functional Rating Scale, change in Revised Amyotrophic Lateral Sclerosis Functional Rating Scale over disease duration and respiratory function. We demonstrate a shift in monocyte subpopulations in patients with amyotrophic lateral sclerosis, with the ratio of classical to non-classical monocytes increased compared with healthy controls. In line with this, patients with greater disease severity, as determined by a lower Revised Amyotrophic Lateral Sclerosis Functional Rating Scale score, had reduced non-classical monocytes. Interestingly, patients with greater bulbar involvement had a reduction in the proportions of classical, intermediate and non-classical monocyte populations. We also revealed several notable associations between myeloid marker expression and clinical features in amyotrophic lateral sclerosis. CD16 expression on neutrophils was increased in patients with greater disease severity and a faster rate of disease progression, whereas HLA-DR expression on all monocyte populations was elevated in patients with greater respiratory impairment. This study demonstrates that patients with amyotrophic lateral sclerosis with distinct clinical features have differential myeloid cell signatures. Identified cell populations and markers may be candidates for targeted mechanistic studies and immunomodulation therapies in amyotrophic lateral sclerosis.
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http://dx.doi.org/10.1093/braincomms/fcaa013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7530830PMC
February 2020

Absence of the C5a Receptor C5aR2 Worsens Ischemic Tissue Injury by Increasing C5aR1-Mediated Neutrophil Infiltration.

J Immunol 2020 11 7;205(10):2834-2839. Epub 2020 Oct 7.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, Queensland 4072, Australia

Neutrophil infiltration to ischemic tissues following reperfusion worsens injury. A key driver of neutrophil recruitment and activation is the complement factor C5a, which signals through two receptors, C5aR1 and C5aR2. In this study, we used a neutrophil-dependent mouse model of intestinal ischemia-reperfusion (IR) injury to investigate the underexplored role of C5aR2 in neutrophil mobilization, recruitment, and disease outcomes. We show that intestinal IR induces rapid neutrophil mobilization along with a concomitant reduction in plasma C5a levels that is driven by both C5aR1 and C5aR2. Intestinal IR in C5aR2 mice led to worsened intestinal damage and increased neutrophil infiltration. Inhibition of C5aR1 signaling in C5aR2 mice with PMX53 prevented neutrophil accumulation and reduced IR pathology, suggesting a key requirement for enhanced neutrophil C5aR1 activation in the absence of C5aR2 signaling. Interestingly, C5aR2 deficiency also reduced circulating neutrophil numbers after IR, as well as following G-CSF-mediated bone marrow mobilization, which was independent of C5aR1, demonstrating that C5aR2 has unique and distinct functions from C5aR1 in neutrophil egress. Despite enhanced tissue injury in C5aR2 IR mice, there were significant reductions in intestinal proinflammatory cytokines, highlighting complicated dual protective/pathogenic roles for C5aR2 in pathophysiology. Collectively, we show that C5aR2 is protective in intestinal IR by inhibiting C5aR1-mediated neutrophil recruitment to the ischemic tissue. This is despite the potentially local pathogenic effects of C5aR2 in increasing intestinal proinflammatory cytokines and enhancing circulating neutrophil numbers in response to mobilizing signals. Our data therefore suggest that this balance between the dual pro- and anti-inflammatory roles of C5aR2 ultimately dictates disease outcomes.
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http://dx.doi.org/10.4049/jimmunol.2000778DOI Listing
November 2020

The Complement C5a-C5aR1 GPCR Axis in COVID-19 Therapeutics.

Trends Immunol 2020 11 23;41(11):965-967. Epub 2020 Sep 23.

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India. Electronic address:

The current pandemic of coronavirus disease (COVID-19) caused by SARS-CoV-2 is a significant global health challenge. A recent study by Carvelli and colleagues now demonstrates the involvement of complement C5a and its receptor C5aR1 in disease progression and suggests that blockade of the C5a-C5aR1 axis may represent a potential therapeutic strategy against COVID-19.
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http://dx.doi.org/10.1016/j.it.2020.09.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510552PMC
November 2020

Acetate protects against intestinal ischemia-reperfusion injury independent of its cognate free fatty acid 2 receptor.

FASEB J 2020 08 17;34(8):10418-10430. Epub 2020 Jun 17.

School of Biomedical Sciences, The University of Queensland, Brisbane, QLD, Australia.

Free fatty acid 2 receptor (FFA2) is highly expressed on neutrophils and, when activated by its cognate ligand acetate, generates potent anti-inflammatory activities. The roles of FFA2 and acetate have not been explored in ischemia-reperfusion injury (IRI). We therefore examined the function of FFA2 and the therapeutic potential of acetate to reduce tissue injury in an acute model of intestinal IRI. The superior mesenteric artery of wild-type (WT) and FFA2 mice was briefly occluded then reperfused following treatment with acetate or vehicle. The absence of FFA2 resulted in intestinal injury similar to that observed in WT mice, indicating a minimal causal role for FFA2 in this model. Acetate treatment to WT mice prior to ischemia profoundly protected the intestine from IRI-induced damage. Amelioration of IRI was also observed, although to a lesser extent, when acetate was administered to FFA2 mice demonstrating that certain protective effects of acetate were FFA2-independent. Remarkably, despite the lack of tissue damage following IRI, acetate-treated mice had markedly increased neutrophil infiltration to the reperfused intestine which was dependent on FFA2. These studies reveal a minimal causal role for FFA2 in intestinal IRI but highlight the novel therapeutic potential for acetate in the amelioration of ischemia-mediated tissue damage.
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http://dx.doi.org/10.1096/fj.202000960RDOI Listing
August 2020

COVID-19: Complement, Coagulation, and Collateral Damage.

J Immunol 2020 09 22;205(6):1488-1495. Epub 2020 Jul 22.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, Queensland 4072, Australia; and

Coronavirus disease of 2019 (COVID-19) is a highly contagious respiratory infection that is caused by the severe acute respiratory syndrome coronavirus 2. Although most people are immunocompetent to the virus, a small group fail to mount an effective antiviral response and develop chronic infections that trigger hyperinflammation. This results in major complications, including acute respiratory distress syndrome, disseminated intravascular coagulation, and multiorgan failure, which all carry poor prognoses. Emerging evidence suggests that the complement system plays a key role in this inflammatory reaction. Indeed, patients with severe COVID-19 show prominent complement activation in their lung, skin, and sera, and those individuals who were treated with complement inhibitors all recovered with no adverse reactions. These and other studies hint at complement's therapeutic potential in these sequalae, and thus, to support drug development, in this review, we provide a summary of COVID-19 and review complement's role in COVID-19 acute respiratory distress syndrome and coagulopathy.
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http://dx.doi.org/10.4049/jimmunol.2000644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7484432PMC
September 2020

Pharmacological characterisation of small molecule C5aR1 inhibitors in human cells reveals biased activities for signalling and function.

Biochem Pharmacol 2020 10 17;180:114156. Epub 2020 Jul 17.

School of Biomedical Sciences, Australia; Queensland Brain Institute, Australia. Electronic address:

The complement fragment C5a is a core effector of complement activation. C5a, acting through its major receptor C5aR1, exerts powerful pro-inflammatory and immunomodulatory functions. Dysregulation of the C5a-C5aR1 axis has been implicated in numerous immune disorders, and the therapeutic inhibition of this axis is therefore imperative for the treatment of these diseases. A myriad of small-molecule C5aR1 inhibitors have been developed and independently characterised over the past two decades, however the pharmacological properties of these compounds has been difficult to directly compare due to the wide discrepancies in the model, read-out, ligand dose and instrumentation implemented across individual studies. Here, we performed a systematic characterisation of the most commonly reported and clinically advanced small-molecule C5aR1 inhibitors (peptidic: PMX53, PMX205 and JPE1375; non-peptide: W545011, NDT9513727, DF2593A and CCX168). Through signalling assays measuring C5aR1-mediated cAMP and ERK1/2 signalling, and β-arrestin 2 recruitment, this study highlighted the signalling-pathway dependence of the rank order of potencies of the C5aR1 inhibitors. Functional experiments performed in primary human macrophages demonstrated the high insurmountable antagonistic potencies for the peptidic inhibitors as compared to the non-peptide compounds. Finally, wash-out studies provided novel insights into the duration of inhibition of the C5aR1 inhibitors, and confirmed the long-lasting antagonistic properties of PMX53 and CCX168. Overall, this study revealed the potent and prolonged antagonistic activities of selected peptidic C5aR1 inhibitors and the unique pharmacological profile of CCX168, which thus represent ideal candidates to fulfil diverse C5aR1 research and clinical therapeutic needs.
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http://dx.doi.org/10.1016/j.bcp.2020.114156DOI Listing
October 2020

Absence of Receptor for Advanced Glycation End Product (RAGE) Reduces Inflammation and Extends Survival in the hSOD1 Mouse Model of Amyotrophic Lateral Sclerosis.

Mol Neurobiol 2020 Oct 16;57(10):4143-4155. Epub 2020 Jul 16.

School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, QLD, 4072, Australia.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron degenerative disease that is without effective treatment. The receptor for advanced glycation end products (RAGE) is a major component of the innate immune system that has been implicated in ALS pathogenesis. However, the contribution of RAGE signalling to the neuroinflammation that underlies ALS neurodegeneration remains unknown. The present study therefore generated SOD1 mice lacking RAGE and compared them with SOD1 transgenic ALS mice in respect to disease progression (i.e. body weight, survival and muscle strength), neuroinflammation and denervation markers in the spinal cord and tibialis anterior muscle. We found that complete absence of RAGE signalling exerted a protective effect on SOD1 pathology, slowing disease progression and significantly extending survival by ~ 3 weeks and improving motor function (rotarod and grip strength). This was associated with reduced microgliosis, cytokines, innate immune factors (complement, TLRs, inflammasomes), and oxidative stress in the spinal cord, and a reduction of denervation markers in the tibialis anterior muscle. We also documented that RAGE mRNA expression was significantly increased in the spinal cord and muscles of preclinical SOD1 and TDP43 models of ALS, supporting a widespread involvement for RAGE in ALS pathology. In summary, our results indicate that RAGE signalling drives neuroinflammation and contributes to neurodegeneration in ALS and highlights RAGE as a potential immune therapeutic target for ALS.
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http://dx.doi.org/10.1007/s12035-020-02019-9DOI Listing
October 2020

A validated quantitative method for the assessment of neuroprotective barrier impairment in neurodegenerative disease models.

J Neurochem 2020 Jul 6. Epub 2020 Jul 6.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, Qld, Australia.

The blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) are highly specialized structures that limit molecule entry from the blood and maintain homeostasis within the central nervous system (CNS). BBB and BSCB breakdown are associated with multiple neurodegenerative diseases. Given the key role of neuroprotective barrier impairment in neurodegeneration, it is important to identify an effective quantitative method to assess barrier integrity in animal models. In this study, we developed and validated a quantitative method for assessing BBB and BSCB integrity using sodium fluorescein, a compound that outperformed other fluorescent dyes. We demonstrated using this method that multiple CNS regions progressively increase in permeability in models of Huntington's disease and amyotrophic lateral sclerosis, whereas biphasic disruption occurred in a mouse model of Alzheimer's disease with disease progression. Collectively, we report a quantitative fluorometric marker with validated reproducible experimental methods that allows the effective assessment of BBB and BSCB integrity in animal models. This method could be useful to further the understanding of the contribution of these neuroprotective barriers to neurodegeneration processes.
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http://dx.doi.org/10.1111/jnc.15119DOI Listing
July 2020

C5aR2 Activation Broadly Modulates the Signaling and Function of Primary Human Macrophages.

J Immunol 2020 08 1;205(4):1102-1112. Epub 2020 Jul 1.

School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, Queensland 4072, Australia

The complement activation fragment C5a is a potent proinflammatory mediator that is increasingly recognized as an immune modulator. C5a acts through two C5a receptors, C5aR1 (C5aR, CD88) and C5aR2 (C5L2, GPR77), to powerfully modify multiple aspects of immune cell function. Although C5aR1 is generally acknowledged to be proinflammatory and immune-activating, the potential roles played by C5aR2 remain poorly defined. Despite studies demonstrating C5aR2 can modulate C5aR1 in human cells, it is not yet known whether C5aR2 functionality is limited to, or requires, C5aR1 activation or influences immune cells more broadly. The present study, therefore, aimed to characterize the roles of C5aR2 on the signaling and function of primary human monocyte-derived macrophages, using a C5aR2 agonist (Ac-RHYPYWR-OH; P32) to selectively activate the receptor. We found that although C5aR2 activation with P32 by itself was devoid of any detectable MAPK signaling activities, C5aR2 agonism significantly dampened C5aR1-, C3aR-, and chemokine-like receptor 1 (CMKLR1)-mediated ERK signaling and altered intracellular calcium mobilization mediated by these receptors. Functionally, selective C5aR2 activation also downregulated cytokine production triggered by various TLRs (TLR2, TLR3, TLR4, and TLR7), C-type lectin receptors (Dectin-1, Dectin-2, and Mincle), and the cytosolic DNA sensor stimulator of IFN genes (STING). Surprisingly, activity at the C-type lectin receptors was particularly powerful, with C5aR2 activation reducing Mincle-mediated IL-6 and TNF-α generation by 80-90%. In sum, this study demonstrates that C5aR2 possesses pleiotropic functions in primary human macrophages, highlighting the role of C5aR2 as a powerful regulator of innate immune function.
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http://dx.doi.org/10.4049/jimmunol.2000407DOI Listing
August 2020

Emerging Insights into the Structure and Function of Complement C5a Receptors.

Trends Biochem Sci 2020 08 10;45(8):693-705. Epub 2020 May 10.

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India. Electronic address:

Complement factor C5a is an integral constituent of the complement cascade critically involved in the innate immune response, and it exerts its functions via two distinct receptors, C5aR1 and C5aR2. While C5aR1 is a prototypical G-protein-coupled receptor (GPCR), C5aR2 lacks functional coupling to heterotrimeric G proteins, although both receptors efficiently recruit β arrestins (βarrs). Here, we discuss the recent studies providing direct structural details of ligand-receptor interactions, and a framework of functional bias in this system, including the differences in terms of structural motifs and transducer coupling. We also discuss the functional analogy of C5aR2 with the atypical chemokine receptors (ACKRs), and highlight the future directions to elucidate the mechanistic basis of the functional divergence of these receptors activated by a common natural agonist.
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http://dx.doi.org/10.1016/j.tibs.2020.04.004DOI Listing
August 2020

The Peripheral Immune System and Amyotrophic Lateral Sclerosis.

Front Neurol 2020 21;11:279. Epub 2020 Apr 21.

Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia.

Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease that is defined by loss of upper and lower motor neurons, associated with accumulation of protein aggregates in cells. There is also pathology in extra-motor areas of the brain, Possible causes of cell death include failure to deal with the aggregated proteins, glutamate toxicity and mitochondrial failure. ALS also involves abnormalities of metabolism and the immune system, including neuroinflammation in the brain and spinal cord. Strikingly, there are also abnormalities of the peripheral immune system, with alterations of T lymphocytes, monocytes, complement and cytokines in the peripheral blood of patients with ALS. The precise contribution of the peripheral immune system in ALS pathogenesis is an active area of research. Although some trials of immunomodulatory agents have been negative, there is strong preclinical evidence of benefit from immune modulation and further trials are currently underway. Here, we review the emerging evidence implicating peripheral immune alterations contributing to ALS, and their potential as future therapeutic targets for clinical intervention.
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http://dx.doi.org/10.3389/fneur.2020.00279DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7186478PMC
April 2020

The potential interplay between energy metabolism and innate complement activation in amyotrophic lateral sclerosis.

FASEB J 2020 06 19;34(6):7225-7233. Epub 2020 Apr 19.

School of Biomedical Sciences, The University of Queensland, St. Lucia, QLD, Australia.

Amyotrophic lateral sclerosis (ALS) is a fatal and rapidly progressing motor neuron disease without effective treatment. Although the precise mechanisms leading to ALS are yet to be determined, there is now increasing evidence implicating the defective energy metabolism and components of the innate immune complement system in the onset and progression of its motor phenotypes. This review will survey the mechanisms by which the energy metabolism and the complement system are altered during the disease progression of ALS and how it can contribute to disease. Furthermore, it will also examine how complement activation can modify the energy metabolism in metabolic disorders, in order to highlight how the complement system and energy metabolism may be linked in ALS.
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http://dx.doi.org/10.1096/fj.201901781DOI Listing
June 2020

Sustained-release ketamine-loaded nanoparticles fabricated by sequential nanoprecipitation.

Int J Pharm 2020 May 4;581:119291. Epub 2020 Apr 4.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, QLD, Australia.

Ketamine in sub-anaesthetic doses is an analgesic adjuvant with a morphine-sparing effect. Co-administration of a strong opioid with an analgesic adjuvant such as ketamine is a potential treatment option, especially for patients with cancer-related pain. A limitation of ketamine is its short in vivo elimination half-life. Hence, our aim was to develop biocompatible and biodegradable ketamine-loaded poly(ethylene glycol) (PEG)-block-poly(lactic-co-glycolic acid) (PLGA) nanoparticles for sustained release. Ketamine-encapsulated single polymer PEG-PLGA nanoparticles and double polymer PEG-PLGA/shellac (SH) nanoparticles with a high drug loading of 41.8% (drug weight/the total weight of drug-loaded nanoparticles) were prepared using a new sequential nanoprecipitation method. These drug-loaded nanoparticles exhibited a sustained-release profile for up to 21 days in vitro and for more than 5 days after intravenous injection in mice. Our study demonstrates that high drug loading and a sustained release profile can be achieved with ketamine-loaded PEG-PLGA nanoparticles prepared using this new nanoprecipitation method.
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http://dx.doi.org/10.1016/j.ijpharm.2020.119291DOI Listing
May 2020

Complement: Bridging the innate and adaptive immune systems in sterile inflammation.

J Leukoc Biol 2020 07 17;108(1):339-351. Epub 2020 Mar 17.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, St Lucia, Brisbane, Queensland, Australia.

The complement system is a collection of soluble and membrane-bound proteins that together act as a powerful amplifier of the innate and adaptive immune systems. Although its role in infection is well established, complement is becoming increasingly recognized as a key contributor to sterile inflammation, a chronic inflammatory process often associated with noncommunicable diseases. In this context, damaged tissues release danger signals and trigger complement, which acts on a range of leukocytes to augment and bridge the innate and adaptive immune systems. Given the detrimental effect of chronic inflammation, the complement system is therefore well placed as an anti-inflammatory drug target. In this review, we provide a general outline of the sterile activators, effectors, and targets of the complement system and a series of examples (i.e., hypertension, cancer, allograft transplant rejection, and neuroinflammation) that highlight complement's ability to bridge the 2 arms of the immune system.
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http://dx.doi.org/10.1002/JLB.3MIR0220-270RDOI Listing
July 2020

Editorial: The Role of Complement in Tumors.

Front Immunol 2020 11;11:139. Epub 2020 Feb 11.

Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, QLD, Australia.

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http://dx.doi.org/10.3389/fimmu.2020.00139DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7027363PMC
March 2021

Preclinical Pharmacokinetics of Complement C5a Receptor Antagonists PMX53 and PMX205 in Mice.

ACS Omega 2020 Feb 30;5(5):2345-2354. Epub 2020 Jan 30.

School of Biomedical Sciences, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia.

The cyclic hexapeptides PMX53 and PMX205 are potent noncompetitive inhibitors of complement C5a receptor 1 (C5aR1). They are widely utilized to study the role of C5aR1 in mouse models, including central nervous system (CNS) disease, and are dosed through a variety of routes of administration. However, a comprehensive pharmacokinetics analysis of these drugs has not been reported. In this study, the blood and CNS pharmacokinetics of PMX53 and PMX205 were performed in mice following intravenous, intraperitoneal, subcutaneous, and oral administration at identical doses. The absorption and distribution of both drugs were rapid and followed a two-compartment model with elimination half-lives of ∼20 min for both compounds. Urinary excretion was the major route of elimination following intravenous dosing with ∼50% of the drug excreted unchanged within the first 12 h. Oral bioavailability of PMX205 was higher than that of PMX53 (23% versus 9%), and PMX205 was also more efficient than PMX53 at entering the intact CNS. In comparison to other routes, subcutaneous administration of PMX205 resulted in high bioavailability (above 90%), as well as prolonged plasma and CNS exposure. Finally, repeated daily oral or subcutaneous administration of PMX205 demonstrated no accumulation of drug in blood, the brain, or the spinal cord, promoting its safety for chronic dosing. These results will be helpful in correlating the desired therapeutic effects of these C5aR1 antagonists with their pharmacokinetic profile. It also suggests that subcutaneous dosing of PMX205 may be an appropriate route of administration for future clinical testing in neurological disease.
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http://dx.doi.org/10.1021/acsomega.9b03735DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017397PMC
February 2020

Low-Fouling Fluoropolymers for Bioconjugation and In Vivo Tracking.

Angew Chem Int Ed Engl 2020 03 3;59(12):4729-4735. Epub 2020 Feb 3.

ARC Centre of Excellence in Convergent Bio-Nano Science and Technology and Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, St Lucia, Queensland, 4072, Australia.

The conjugation of hydrophilic low-fouling polymers to therapeutic molecules and particles is an effective approach to improving their aqueous stability, solubility, and pharmacokinetics. Recent concerns over the immunogenicity of poly(ethylene glycol) has highlighted the importance of identifying alternative low fouling polymers. Now, a new class of synthetic water-soluble homo-fluoropolymers are reported with a sulfoxide side-chain structure. The incorporation of fluorine enables direct imaging of the homopolymer by F MRI, negating the need for additional synthetic steps to attach an imaging moiety. These self-reporting fluoropolymers show outstanding imaging sensitivity and remarkable hydrophilicity, and as such are a new class of low-fouling polymer for bioconjugation and in vivo tracking.
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http://dx.doi.org/10.1002/anie.201914119DOI Listing
March 2020

Complement dysregulation in the central nervous system during development and disease.

Semin Immunol 2019 10 7;45:101340. Epub 2019 Nov 7.

School of Biomedical Sciences, Faculty of Medicine, The University of Queensland, Brisbane, Australia. Electronic address:

The complement cascade is an important arm of the immune system that plays a key role in protecting the central nervous system (CNS) from infection. Recently, it has also become clear that complement proteins have fundamental roles in the developing and aging CNS that are distinct from their roles in immunity. During neurodevelopment, complement signalling is involved in diverse processes including neural tube closure, neural progenitor proliferation and differentiation, neuronal migration, and synaptic pruning. In acute neurotrauma and ischamic brain injury, complement drives inflammation and neuronal death, but also neuroprotection and regeneration. In diseases of the aging CNS including dementias and motor neuron disease, chronic complement activation is associated with glial activation, and synapse and neuron loss. Proper regulation of complement is thus essential to allow for an appropriately developed CNS and prevention of excessive damage following neurotrauma or during neurodegeneration. This review provides a comprehensive overview of the evidence for functional roles of complement in brain formation, and its dysregulation during acute and chronic disease. We also provide working models for how complement can lead to neurodevelopmental disorders such as schizophrenia and autism, and either protect, or propagate neurodegenerative diseases including Alzheimer's disease and amyotrophic lateral sclerosis.
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http://dx.doi.org/10.1016/j.smim.2019.101340DOI Listing
October 2019

Complement C5a Induces Renal Injury in Diabetic Kidney Disease by Disrupting Mitochondrial Metabolic Agility.

Diabetes 2020 01 17;69(1):83-98. Epub 2019 Oct 17.

Department of Diabetes, Central Clinical School, Alfred Medical Research and Education Precinct, Monash University, Melbourne, Victoria, Australia

The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD.
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http://dx.doi.org/10.2337/db19-0043DOI Listing
January 2020
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