C4BP-IgM protein as a novel therapeutic approach to treat Neisseria gonorrhoeae infections

• Text
• PDF

Abstract

Gonorrhea is a sexually transmitted infection with 87 million new cases per year globally. Increasing antibiotic resistance has severely limited treatment options. A mechanism that Neisseria gonorrhoeae uses to evade complement attack is binding of the complement inhibitor C4b-binding protein (C4BP). We screened 107 PorB1a and 83 PorB1b clinical isolates randomly selected from a Swedish strain collection over the last 10 years and noted that 96/107 (89.7%) PorB1a and 16/83 (19.3%) PorB1b bound C4BP; C4BP binding significantly correlated with the ability to evade complement-dependent killing (r = 0.78; p<0.0001). We designed two chimeric proteins that fused C4BP domains to the backbone of immunoglobulins IgG or IgM (C4BP-IgG; C4BP-IgM) with the aim of enhancing complement activation and killing of gonococci. Both proteins bound gonococci (Kd C4BP-IgM = 2.4 nM; Kd C4BP-IgG 981 nM), but only hexameric C4BP-IgM efficiently out-competed heptameric C4BP from bacterial surface resulting in enhanced complement deposition and bacterial killing. Furthermore, C4BP-IgM significantly attenuated the duration and burden of colonization of two C4BP-binding gonococcal isolates, but not a C4BP non-binding strain in the mouse vaginal colonization model using human factor H/C4BP transgenic mice. Our pre-clinical data present C4BP-IgM as an adjunctive to conventional antimicrobials for the treatment of gonorrhea.

Authors

Serena Bettoni, Jutamas Shaughnessy, Karolina Maziarz, David Ermert, Sunita Gulati, Bo Zheng, Matthias Mörgelin, Susanne Jacobsson, Kristian Riesbeck, Magnus Unemo, Sanjay Ram, Anna M. Blom

Uncoupled turnover disrupts mitochondrial quality control in diabetic retinopathy

• Text
• PDF

Abstract

Mitochondrial quality control (MQC) is crucial for regulating central nervous system homeostasis and its disruption has been implicated in the pathogenesis of some of the most common neurodegenerative diseases. In healthy tissues, the maintenance of MQC depends upon an exquisite balance between mitophagy (removal of damaged mitochondria by autophagy) and biogenesis (de-novo synthesis of mitochondria). Here, we show that mitophagy is disrupted in diabetic retinopathy (DR) and decoupled from mitochondrial biogenesis during the progression of the disease. Diabetic retinas from human post-mortem donors and experimental mice exhibit a net loss of mitochondrial contents during the early stages of the disease process. Using novel diabetic mitophagy-reporter mice (mitoQC-Ins2Akita) alongside pMitoTimer (a molecular clock to address mitochondrial-age dynamics), we demonstrate that mitochondrial loss arose due to an inability of mitochondrial biogenesis to compensate for diabetes-exacerbated mitophagy. However, as diabetes duration increases, Pink1-dependent mitophagy deteriorates, leading to the build-up of mitochondria primed for degradation in DR. Impairment of mitophagy during prolonged diabetes is linked with the development of retinal senescence, a phenotype that blunted hyperglycaemia-induced mitophagy in mitoQC primary Müller cells. Our findings suggest that normalizing mitochondrial turnover may preserve MQC and provide novel therapeutic options for the management of DR-associated complications.

Authors

Jose R. Hombrebueno, Lauren Cairns, Louise R. Dutton, Timothy J. Lyons, Derek P. Brazil, Paul Moynagh, Tim M. Curtis, Heping Xu

Mevastatin promotes healing by targeting Caveolin-1 to restore EGFR signaling

• Text
• PDF

Abstract

Diabetic foot ulcers (DFUs) are a life-threatening disease that often result in lower limb amputations and a shortened lifespan. Current treatment options are limited and often not efficacious, raising the need for new therapies. To investigate the therapeutic potential of topical statins to restore healing in patients with DFUs, we performed next generation sequencing on mevastatin-treated primary human keratinocytes. We found that mevastatin activated and modulated the EGF signaling to trigger an anti-proliferative and pro-migratory phenotype, suggesting that statins may shift DFUs from a hyper-proliferative phenotype to a pro-migratory phenotype in order to stimulate healing. Furthermore, mevastatin induced a migratory phenotype in primary human keratinocytes through EGF-mediated activation of Rac1, resulting in actin cytoskeletal reorganization and lamellipodia formation. Interestingly, the EGF receptor is downregulated in tissue biopsies from patients with DFUs. Mevastatin restored EGF signaling in DFUs through disruption of caveolae to promote keratinocyte migration, which was confirmed by caveolin-1 (Cav1) overexpression studies. We conclude that topical statins may have considerable therapeutic potential as a treatment option for patients with DFUs and offer an effective treatment for chronic wounds that can be rapidly translated to clinical use.

Authors

Andrew P. Sawaya, Ivan Jozic, Rivka C. Stone, Irena Pastar, Andjela N. Egger, Olivera Stojadinovic, George D. Glinos, Robert S. Kirsner, Marjana Tomic-Canic

Proteasome inhibition preserves longitudinal growth of denervated muscle and prevents neonatal neuromuscular contractures

• Text
• PDF

Abstract

Muscle contractures are a prominent and disabling feature of many neuromuscular disorders, including the two most common forms of childhood neurologic dysfunction: neonatal brachial plexus injury (NBPI) and cerebral palsy (CP). There are currently no treatment strategies to directly alter the contracture pathology, as the pathogenesis of these contractures is unknown. We previously showed in a mouse model of NBPI that contractures result from impaired longitudinal muscle growth. Current presumed explanations for growth impairment in contractures focus on the dysregulation of muscle stem cells (MuSCs), which differentiate and fuse to existing myofibers during growth, as this process has classically been thought to control muscle growth during the neonatal period. Here, we demonstrate in a mouse model of NBPI that denervation does not prevent myonuclear accretion and that reduction of myonuclear number has no effect on functional muscle length or contracture development, providing definitive evidence that altered myonuclear accretion is not a driver of neuromuscular contractures. In contrast, we observed elevated levels of protein degradation in NBPI muscle, and we demonstrate that contractures can be pharmacologically prevented with the proteasome inhibitor, bortezomib. These studies provide the first strategy to prevent neuromuscular contractures by correcting the underlying deficit in longitudinal muscle growth.

Authors

Sia Nikolaou, Alyssa A.W. Cramer, Liangjun Hu, Qingnian Goh, Douglas P. Millay, Roger Cornwall

EPHB2 carried on small extracellular vesicles induces tumor angiogenesis via activation of ephrin reverse signaling

• Text
• PDF

Abstract

Angiogenesis is a key process that allows nutrient uptake and cellular trafficking and is co-opted in cancer to enable tumor growth and metastasis. Recently, extracellular vesicles (EVs) have been shown to promote angiogenesis; however, it is unclear what unique features EVs contribute to the process. Here, we studied the role of EVs derived from head and neck squamous cell carcinoma (HNSCC) in driving tumor angiogenesis. Small EVs (SEVs), in the size range of exosomes (50-150 nm), induced angiogenesis both in vitro and in vivo. Proteomic analysis of HNSCC SEVs revealed the cell-cell signaling receptor EPHB2 as a promising candidate cargo to promote angiogenesis. Analysis of TCGA RNA-Seq and patient tissue microarray data further identified EPHB2 overexpression in HNSCC tumors to be associated with poor patient prognosis and tumor angiogenesis, especially in the context of overexpression of the exosome secretion regulator cortactin. Functional experiments revealed that EPHB2 expression in SEVs regulates angiogenesis both in vitro and in vivo and that EPHB2 carried by SEVs stimulates ephrin-B reverse signaling, inducing STAT3 phosphorylation. A STAT3 inhibitor greatly reduced SEV-induced angiogenesis. These data suggest a novel model in which EVs uniquely promote angiogenesis by transporting Eph transmembrane receptors to non-adjacent endothelial cells to induce ephrin reverse signaling.

Authors

Shinya Sato, Suhas Vasaikar, Adel Eskaros, Young Kim, James S. Lewis, Bing Zhang, Andries Zijlstra, Alissa M. Weaver

ER stress and Rho kinase activation underlie the vasculopathy of CADASIL

• Text
• PDF

Abstract

CADASIL leads to premature stroke and vascular dementia. Mechanism-specific therapies for this aggressive cerebral small vessel disease are lacking. CADASIL is caused by NOTCH3 mutations that influence vascular smooth muscle cell (VSMC) function through unknown processes. We investigated molecular mechanisms underlying the vasculopathy in CADASIL focusing on ER stress and RhoA/Rho kinase (ROCK). Peripheral small arteries and VSMCs were isolated from gluteal biopsies of CADASIL patients and mesentery of TgNotch3R169C mice (CADASIL model). CADASIL vessels exhibited impaired vasorelaxation, blunted vasoconstriction and hypertrophic remodelling. Expression of NOTCH3 and ER stress target genes was amplified and ER stress response, Rho kinase activity, superoxide production and cytoskeletal-associated protein phosphorylation were increased in CADASIL, processes associated with Nox5 upregulation. Aberrant vascular responses and signalling in CADASIL were ameliorated by inhibitors of Notch3 (gamma-secretase inhibitor), Nox5 (mellitin), ER stress (4-PBA) and ROCK (fasudil). Observations in human CADASIL were recapitulated in TgNotch3R169C mice. These findings indicate that vascular dysfunction in CADASIL involves ER stress/ROCK interplay driven by Notch3-induced Nox5 activation and that NOTCH3 mutation-associated vascular pathology, typical in cerebral vessels, also manifests peripherally. We define Notch3-Nox5/ERstress/ROCK signaling as a novel putative mechanism-specific target and suggest that peripheral artery responses may be an accessible biomarker in CADASIL.

Authors

Karla B. Neves, Adam P. Harvey, Fiona Moreton, Augusto C. Montezano, Francisco J. Rios, Rhéure Alves-Lopes, Aurelie Nguyen Dinh Cat, Paul Rocchiccioli, Christian Delles, Anne Joutel, Keith Muir, Rhian M. Touyz

Insulin is produced in choroid plexus and its release is regulated by serotonergic signaling

• Text
• PDF

Abstract

The choroid plexus (ChP) is a highly vascularized tissue found in the brain ventricles, with an apical epithelial cell layer surrounding fenestrated capillaries. It is responsible for the production of most of the cerebrospinal fluid (CSF) in the ventricular system, subarachnoid space, and central canal of the spinal cord, while also constituting the blood-CSF barrier (BCSFB). In addition, epithelial cells of the choroid plexus (EChP) synthesize neurotrophic factors and other signaling molecules that are released into the CSF. Here we show that insulin is produced in EChP of mice and humans, and its expression and release are regulated by serotonin. Insulin mRNA and immune-reactive protein, including C-peptide, are present in EChP, as detected by several experimental approaches, and in much higher levels than any other brain region and non-pancreatic peripheral tissues. Moreover, insulin is produced in primary cultured mouse EChP, and its release, albeit Ca2+-sensitive, is not regulated by glucose. Instead, activation of the 5HT2C receptor by serotonin treatment led to activation of IP3-sensitive channels and Ca2+ mobilization from intracellular storage, leading to insulin secretion. In vivo depletion of brain serotonin in the dorsal raphe nucleus negatively affected insulin expression in the ChP, suggesting an endogenous modulation of ChP insulin by serotonin. Therefore, for the first time to our knowledge, here we show that insulin is produced by EChP in the brain, and its release is modulated at least by serotonin, and not glucose.

Authors

Caio Henrique Mazucanti, Qing-Rong Liu, Doyle Lang, Nicholas Huang, Jennifer F. O’Connell, Simonetta Camandola, Josephine M. Egan

Mitophagy dependent macrophage reprogramming protects against kidney fibrosis

• Text
• PDF

Abstract

Mitophagy, by maintaining mitochondrial quality control, plays a key role in maintaining kidney function and is impaired in pathologic states. Macrophages are well-known for their pathogenic role in kidney fibrosis. Here, we report that PINK1/Parkin-mediated mitophagy in macrophages is compromised in experimental and human kidney fibrosis. We demonstrate downregulation of mitophagy regulators, mitofusin-2 (MFN2) and Parkin, downstream of PINK1 in kidney fibrosis. Loss of either Pink1 or Prkn promoted renal extracellular matrix accumulation and frequency of profibrotic/M2 macrophages. Pink1-/- or Prkn-/- bone-marrow-derived macrophages (BMDMs) showed enhanced expression of rictor. Mitochondria from TGF-β1-treated Pink1-/- BMDMs exhibited increased superoxide levels, and reduced respiration and ATP production. In addition, mitophagy in macrophages involves PINK1-mediated phosphorylation of downstream MFN2 and MFN2-facilitated recruitment of Parkin to damaged mitochondria, and macrophage-specific deletion of Mfn2 aggravates kidney fibrosis. Moreover, mitophagy regulators were downregulated in human CKD kidney and TGF-β1-treated human renal macrophages, whereas Mdivi1 treatment suppressed mitophagy mediators and promoted fibrotic response. Taken together, our study is the first to demonstrate that macrophage mitophagy plays a protective role against kidney fibrosis via regulating PINK1/MFN2/Parkin-mediated pathway.

Authors

Divya Bhatia, Kuei-Pin Chung, Kiichi Nakahira, Edwin Patino, Michelle C. Rice, Lisa K. Torres, Thangamani Muthukumar, Augustine M.K. Choi, Oleh M. Akchurin, Mary E. Choi

Prelamin A mediates inflammation in dilated and HIV associated cardiomyopathies

• Text
• PDF

Abstract

Cardiomyopathies are complex heart muscle diseases that can be inherited or acquired. Dilated cardiomyopathy can result from mutations in LMNA, encoding the nuclear intermediate filament proteins lamin A/C. Some LMNA mutations lead to accumulation of the lamin A precursor, prelamin A, which is disease causing in a number of tissues yet its impact upon the heart is unknown. Here we discovered myocardial prelamin A accumulation occurred in a case of dilated cardiomyopathy and show that a novel mouse model of cardiac specific prelamin A accumulation exhibited a phenotype consistent with ‘inflammatory cardiomyopathy’ which we observed to be similar to HIV associated cardiomyopathy, an acquired disease state. Numerous HIV protease therapies are known to inhibit ZMPSTE24, the enzyme responsible for prelamin A processing, and we confirmed that accumulation of prelamin A occurred in HIV+ patient cardiac biopsies. These findings: (1) confirm a unifying pathological role for prelamin A common to genetic and acquired cardiomyopathies; (2) have implications for the management of HIV patients with cardiac disease suggesting protease inhibitors should be replaced with alternative therapies i.e. non-nucleoside reverse transcriptase inhibitors; and (3) suggest that targeting inflammation may be a useful treatment strategy for certain forms of inherited cardiomyopathy.

Authors

Daniel Brayson, Andrea Frustaci, Romina Verardo, Cristina Chimenti, Matteo Antonio Russo, Robert Hayward, Sadia Munir Ahmad, Gema Vizcay-Barrena, Andrea Protti, Peter S. Zammit, Cristobal G. dos Remedios, Elisabeth Ehler, Ajay M. Shah, Catherine M. Shanahan

Keratinocyte-derived IκBζ drives psoriasis and associated systemic inflammation

• Text
• PDF

Abstract

The transcriptional activator IκBζ is a key regulator of psoriasis, but which cells mediate its pathogenic effect remains unknown. Here we found that IκBζ expression in keratinocytes triggers not only skin lesions, but also systemic inflammation in mouse psoriasis models. Specific depletion of IκBζ in keratinocytes was sufficient to suppress the induction of imiquimod- or IL-36-mediated psoriasis. Moreover, IκBζ ablation in keratinocytes prevented the onset of psoriatic lesions and systemic inflammation in keratinocyte-specific IL-17A transgenic mice. Mechanistically, this psoriasis protection was mediated by the fact that IκBζ deficiency in keratinocytes abrogated the induction of specific pro-inflammatory target genes, including Cxcl5, Cxcl2, Csf2 and Csf3, in response to IL-17A or IL-36. These IκBζ-dependent genes trigger the generation and recruitment of neutrophils and monocytes that are needed for skin inflammation. Consequently, our data uncover a surprisingly pivotal role of keratinocytes and keratinocyte-derived IκBζ as key mediators of psoriasis and psoriasis-related systemic inflammation.

Authors

Sebastian Lorscheid, Anne Müller, Jessica Löffler, Claudia Resch, Philip Bucher, Florian C. Kurschus, Ari Waisman, Knut Schäkel, Stephan Hailfinger, Klaus Schulze-Osthoff, Daniela Kramer