Rho-inhibiting C2IN-C3 fusion toxin inhibits chemotactic recruitment of human monocytes ex vivo and in mice in vivo

Tobias Martin,Anne Rittlinger,Julian Schneider,Markus Huber-Lang,Holger Barth,Annette Palmer,Stephanie Denk,Stephan Paschke,Mona Vogel,Hartmut Geiger,Amelie Möglich,Ina Felix,Christina Förtsch,Lena Notbohm

doi:10.1007/s00204-017-2058-y

Tobias Martin, Anne Rittlinger + Show 12 more

Open Access

https://doi.org/10.1007/s00204-017-2058-y

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Abstract

Bacterial protein toxins became valuable molecular tools for the targeted modulation of cell functions in experimental pharmacology and attractive therapeutics because of their potent and specific mode of action in human cells. C2IN-C3lim, a recombinant fusion toxin (~50 kDa) of the Rho-inhibiting C3lim from Clostridium (C.) limosum and a non-toxic portion of the C. botulinum C2 toxin (C2IN), is selectively internalized into the cytosol of monocytic cells where C3lim specifically ADP-ribosylates Rho A and -B, thereby inhibiting Rho-mediated signaling. Thus, we hypothesized that these unique features make C2IN-C3lim an attractive molecule for the targeted pharmacological down-regulation of Rho-mediated functions in monocytes. The analysis of the actin structure and the Rho ADP-ribosylation status implied that C2IN-C3lim entered the cytosol of primary human monocytes from healthy donors ex vivo within 1 h. Moreover, it inhibited the fMLP-induced chemotaxis of human monocytes in a Boyden chamber model ex vivo. Similarly, in a 3-dimensional ex vivo model of extravasation, single cell analysis revealed that C2IN-C3lim-treated cells were not able to move. In a clinically relevant mouse model of blunt chest trauma, the local application of C2IN-C3lim into the lungs after thorax trauma prevented the trauma-induced recruitment of monocytes into the lungs in vivo. Thus, C2IN-C3lim might be an attractive lead compound for novel pharmacological strategies to avoid the cellular damage response caused by monocytes in damaged tissue after trauma and during systemic inflammation. The results suggest that the pathophysiological role of clostridial C3 toxins might be a down-modulation of the innate immune system.

Highlights

We and others identified monocytes/macrophages as target cells for the C3 protein toxins (~25 kDa) from Clostridium (C.) botulinum (C3bot1) and C. limosum (C3lim) (Fahrer et al 2010; Dmochewitz et al 2013; Christow et al 2013; Rotsch et al 2012; Tautzenberger et al 2013; Rohrbeck et al 2014)
The results suggest an immunosuppressive mode of action of C3 toxins and identified the recombinant fusion toxin C2INC3lim as an attractive candidate for the targeted pharmacological down-modulation of monocyte recruitment and accumulation in damaged tissues/organs which might be detrimental after traumatic injury
Based on the results that C3bot1 down-modulates the migration of macrophages in vitro (Wheeler and Ridley 2007; Rotsch et al 2012) and that recombinant C2 toxin (C2IN)-C3lim acts significantly stronger on RAW macrophages and derived osteoclasts than the natural C3bot1 (Tautzenberger et al 2013), we investigated whether C2IN-C3lim is more potent regarding the inhibition of macrophage migration

Summary

Introduction

We and others identified monocytes/macrophages as target cells for the C3 protein toxins (~25 kDa) from Clostridium (C.) botulinum (C3bot1) and C. limosum (C3lim) (Fahrer et al 2010; Dmochewitz et al 2013; Christow et al 2013; Rotsch et al 2012; Tautzenberger et al 2013; Rohrbeck et al 2014). The clostridial C3 toxins catalyze the covalent transfer of an ADP-ribose moiety from NAD onto Asn-41 of the GTPases Rho A, -B, -C (Aktories and Frevert 1987; Aktories et al 1995; Just et al 1992; Han et al 2001; for review see Aktories 2011), which inhibits the Rho-mediated signal transduction in mammalian cells (Rubin et al 1988; Chardin et al 1989; Etienne-Manneville and Hall 2002). The inhibition of Rho-signaling, i.e. by C3 toxin, results in reorganization of the actin cytoskeleton associated with an altered cell morphology and the Rho/actin-mediated cellular processes are inhibited (for review see Vogelsgesang et al 2007)

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