Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer–CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies

The MT1 agonists Ramelteon and Agomelatine strongly promote NMJ structural recovery after envenoming with <i>B</i>. <i>caeruleus</i>.

Soleus (A) and gastrocnemius (D) muscles were processed for indirect immunofluorescence using fluorescent α-BTX to stain post-synaptic AChRs (red), and anti-VAMP1 antibodies to identify the pre-synaptic compartment (green). Asterisks identify degenerated NMJs while arrows partial innervated ones. Scale bars: 50 μm (10 μm in magnification). Quantification of degenerated, regenerated and partial innervated NMJs in soleus (B) and gastrocnemius (E) muscles. N = 3, 40 NMJs analyzed/muscle. 2way ANOVA, interaction source of Variation ***p < 0,001, ****p <0,0001 in B, **p < 0,001, ****p <0,0001 in B and E. C and F Percentage of NMJ occupancy, which represents the overlap between pre- and post-synaptic markers. One way ANOVA *p<0,05, **p<0,01.</p

Structural recovery of respiratory NMJs after poisoning by <i>B</i>. <i>caeruleus</i> venom is strongly stimulated by Ramelteon or Agomelatine.

Pectoral (A) and intercostal (D) muscles were processed for indirect immunofluorescence using fluorescent α-BTx to stain post-synaptic AChRs (red), and anti-VAMP1 antibodies to identify the pre-synaptic compartment (green). Asterisks identify degenerated NMJs while arrows partial innervated ones. Scale bars: 50 μm (10 μm in magnification). Quantification of degenerated, regenerated and partial innervated NMJs in pectoral (B) and intercostal (E) muscles. N = 3, 40 NMJs analyzed/muscle. 2way ANOVA, interaction source of Variation *p (TIF)</p

<i>Bungarus caeruleus</i> venom causes an acute and reversible degeneration of motor axon terminals at the soleus NMJs and induces the expression of melatonin receptor type 1 (MT1) in Perisynaptic Schwann cells.

A) B. caeruleus venom was injected intramuscularly (i.m.) in the hind limb of anesthetized mice, and regeneration was followed via electrophysiological recordings of the Evocked Junction Potentials (EJPs) at 24h, 96h and 168h after intoxication. Each bar represents mean ± SD. N = 4 animals (Ctrl), N = 3 animals (24h, 96h, 168h), 15 fibres/animal. ****pB. caeruleus at different time points (0, 24 h, 48 h, 96 h, 168 h), and stained with specific antibodies. Motor axon terminals are identified by Neurofilament heavy chain (NF-H, white), PSCs by endogenous GFP (green) and MT1 (red) by a specific monoclonal antibody [58]. Scale bars: 10 μm.</p

The MT1 agonists Ramelteon and Agomelatine strongly promote recovery of function of the NMJ after injection of <i>B</i>. <i>caeruleus</i> venom.

A) Schematic representation of the technique employed to measure the Evoked Junctional Potentials (EJPs), Created with BioRender.com. B) EJPs of soleus muscles 96 hours post injection of B. caeruleus venom in the hind limb of mice treated daily with melatonin (Mel), Ramelteon (Ram), or Agomelatine (Agom). Each bar represents the mean of the EJP amplitude ± SD, at least N = 4, number of analyzed fibers: 15/mice, One way ANOVA ****p B. caeruleus venom in the absence or presence of Ramelteon and Agomelatine. N = 4, One-way ANOVA *p< 0.05. E-F), Representative EJP (E) and CMAP (F) traces of control and treated muscles w/wo Ramelteon or Agomelatine.</p

Excel spreadsheet containing, in separate sheets, the underlying numerical data for Figs 1A, 2B, 2D, 3B, 3C, 3D, 3E, 3F, 4B, 4E, 4F.

Excel spreadsheet containing, in separate sheets, the underlying numerical data for Figs 1A, 2B, 2D, 3B, 3C, 3D, 3E, 3F, 4B, 4E, 4F.</p

Chemical formulas of Melatonin, Ramelteon and Agomelatine.

Chemical formulas of Melatonin, Ramelteon and Agomelatine.</p

Recovery of normal respiration after poisoning with <i>B</i>. <i>caeruleus</i> venom is strongly stimulated by Ramelteon or Agomelatine.

A) Schematic representation of the technique of measurement employed to calculate the Ventilation Index, Created with BioRender.com. B) Quantitative ventilation assay was performed by measuring the peak area (mV/ms) of consecutive peaks within 20 seconds of trace. The average area was multiplied by the frequency within the time period considered (±SEM). The t = 0 was detected before animals’ envenomation, and animals with similar ventilation values were kept to start the experiments. Each group of envenomated animals consisted of N = 4 mice. One-way ANOVA **p < 0,001, ****p <0,001 ****p <0,0001. C) Diaphragm muscles were processed for indirect immunofluorescence using fluorescent α-BTX to stain post-synaptic AChRs (red), and anti-VAMP1 antibodies to identify the pre-synaptic compartment (green). Asterisks identify degenerated NMJs while arrows partial innervated ones. Scale bars: 50 μm (10 μm in magnification). D) Quantification of degenerated, regenerated and partial innervated NMJs. N = 3, 40 NMJs analyzed/muscle. 2way ANOVA, interaction source of Variation *p < 0,05, **p <0,01. E) Percentage of NMJ occupancy, which represents the overlap between pre- and post-synaptic elements. One way ANOVA *p < 0,05, **p <0,01.</p

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<p>Soleus muscles of mice treated with BoNT/C-wt and used for the analysis of EJPs of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006567#ppat.1006567.g005" target="_blank">Fig 5B</a> were fixed immediately after the electrophysiological recordings and stained for <b>(A)</b> cleaved SNAP-25 (SNAP-25_c) or <b>(B)</b> Syntaxin-1A/1B (Stx-1A/1B), both shown in red. NMJs were spotted with α-Bungarotoxin (α-BTX, in green). The first row of panels represents the staining of a control muscle. Scale bar, 10 μm.</p

Botulinum neurotoxin C mutants reveal different effects of syntaxin or SNAP-25 proteolysis on neuromuscular transmission

<div><p>Botulinum neurotoxin serotype C (BoNT/C) is a neuroparalytic toxin associated with outbreaks of animal botulism, particularly in birds, and is the only BoNT known to cleave two different SNARE proteins, SNAP-25 and syntaxin. BoNT/C was shown to be a good substitute for BoNT/A1 in human dystonia therapy because of its long lasting effects and absence of neuromuscular damage. Two triple mutants of BoNT/C, namely BoNT/C <i>S51T/R52N/N53P</i> (BoNT/C α-51) and BoNT/C <i>L200W/M221W/I226W</i> (BoNT/C α-3W), were recently reported to selectively cleave syntaxin and have been used here to evaluate the individual contribution of SNAP-25 and syntaxin cleavage to the effect of BoNT/C <i>in vivo</i>. Although BoNT/C α-51 and BoNT/C α-3W toxins cleave syntaxin with similar efficiency, we unexpectedly found also cleavage of SNAP-25, although to a lesser extent than wild type BoNT/C. Interestingly, the BoNT/C mutants exhibit reduced lethality compared to wild type toxin, a result that correlated with their residual activity against SNAP-25. In spite of this, a local injection of BoNT/C α-51 persistently impairs neuromuscular junction activity. This is due to an initial phase in which SNAP-25 cleavage causes a complete blockade of neurotransmission, and to a second phase of incomplete impairment ascribable to syntaxin cleavage. Together, these results indicate that neuroparalysis of BoNT/C at the neuromuscular junction is due to SNAP-25 cleavage, while the proteolysis of syntaxin provides a substantial, but incomplete, neuromuscular impairment. In light of this evidence, we discuss a possible clinical use of BoNT/C α-51 as a botulinum neurotoxin endowed with a wide safety margin and a long lasting effect.</p></div