Modulation of cellular and molecular components of innate immunity by a polysaccharide derivative as a strategy against virus infections

Despite numerous therapeutic achievements, infectious diseases, such as viral infections, remain a continuous hazard to human health, in part due to emergence or re-emergence of new and hazardous viruses or the lack of appropriate vaccines or prophylactic or post-exposure therapies. Successful elimination of infectious viruses highly depends on the dynamic interplay between leukocytes and proteins of the fast-responding broad-spectrum innate and more slowly and highly specific adaptive immune system. Extensive research efforts during the last decade demonstrated the processes of virus recognition and innate defense signaling by host cells as essential steps that shape the overall immune response to virus infections. As a result, insight into general molecular and cellular mechanisms of innate immunity together with comprehensive understanding of the particular contribution of a specific leukocyte subset within a given virus infection, are critical to understand viral pathogenesis and immunopathology. Furthermore, exploring the mechanisms by which the innate immune system can be triggered to sustain an effective adaptive immune response may pave the way to new therapy and vaccine strategies.COAM (chlorite-oxidized oxyamylose) is a polyanionic polysaccharide derivative which has been synthesized at the Rega Institute and has been known for many years as an antiviral agent. Its broad-spectrum antiviral potency has been demonstrated in lethal virus infection models, such as influenza virus and herpes simplex virus-induced and mengovirus-induced encephalitis. A thorough comprehension about its mechanism of action remained unexplored, though, it was initially suggested that COAM modulates the immune response by acting as an antiviral interferon (IFN) inducer. In the framework of this doctoral research our principal aim was to elucidate which molecules and cells of the innate immune system were responsible for the antiviral resistance induced by COAM in vivo, and to gain insight into the impact of pharmacological modulation of antiviral immunity on the outcome of infection. Throughout this project, a mouse picornavirus, i.e. mengovirus, which induces fatal meningoencephalitis, was used as a model to study the protective mechanisms of COAM on acute lethal virus infection.In a first part of this doctoral thesis, we investigated direct and indirect antiviral mechanisms of COAM in vitro, and attempted to draw a comparison with the dsRNA mimetic polyriboinosinic-polyribocytidylic acid or poly(I:C), which is a well-known antiviral interferon (IFN) inducer. Although COAM and poly(I:C) share a polyanionic nature, their mode of action in vitro relies on different mechanisms. In comparison with a strong IFN induction and mRNA upregulation of the helicases RIG-I and MDA-5 by poly(I:C), COAM did not enhance IFN-a or -ß and IFN-inducible RNA helicases in mouse fibroblastoid cells. Instead, COAM inhibited virus entry by blocking the attachment to the cells. These results suggest that COAM can alter the outcome of in vitro infection, not by IFNinduction and in turn modifying the cellular antiviral state, but through inhibition of virus entry into cells. However, although these data point to virus attachment to the cell membrane as the target of antiviral action, this viral entry-inhibiting effect was rather weak, suggesting that this effect is unlikely to fully account for the previously observed potent in vivo antiviral protection by COAM.Secondly, possible cellular and molecular mechanisms of innate immunity that might underlie the observed antiviral protection by COAM in vivo were further explored. The COAM mixture was fractionated and prophylactic intraperitoneal treatment of mice with COAM polymers of high molecular weight resulted in a conversion from 100% lethal viral encephalitis, caused by mengovirus infection, to an overall survival rate of 93%, without obvious clinical sequelae. COAM induced a profound peritoneal influx of neutrophil granulocytes together with an activation of macrophages, which correlated with decreased viral titers in peritoneum, blood and target organs, and an annihilation of cerebral induction of proinflammatory cytokines. Moreover, COAM was able to interact with the chemokine system. Mouse granulocyte chemotactic protein-2 (GCP-2)/CXCL6, a potent neutrophil attracting chemokine, was found to bind COAM. In addition, COAM also induced mouse GCP-2 in vitro and in vivo. The antiviral effects of COAM decreased significantly in mice depleted of neutrophils and macrophages, which demonstrated that these cells co-determined the antiviral resistance induced by COAM. All together, these results established that COAM rescued mice from acute and lethal mengovirus infection by recruiting antiviral leukocytes to the site of infection, i.e. the peritoneum. These findings reinforce the role of neutrophils and macrophages as key effector cells that can be manipulated toward antiviral defense.In a last part, the concept that COAM modulates the migration of leukocytes in vivo was further explored and extended to interactions with chemokines other than GCP-2. Chemokines are central players in coordinating directional and selective leukocyte migration into tissues. COAM induced the transcriptional program of various mouse CXC and CC chemokines in vitro and also in peritoneal leukocytes upon injection of mice with COAM. In addition, COAM was able to directly interact with chemokines through binding. In particular, efficient binding was observed for the neutrophil chemokines KC/CXCL1 and MIP-2/CXCL2, as well as the monocyte chemoattractant RANTES/CCL5 and IP-10/CXCL10, the latter being chemotactic for monocytes, activated T cells and natural killer cells. Some degree of binding specificity was detected as no binding interactions were observed for MCP-1/CCL2 and MIP-1a/CCL3. Binding affinities of interacting chemokines for COAM were all in the nM-range and were similar to those observed for the glycosaminoglycan heparan sulphate, which represents an endogenous chemokine-binding molecule responsible for sustaining chemotactic gradients across vesselwalls and into the extracellular matrix and tissues. Although COAM did not display chemotactic activity by itself, the chemokine binding feature of COAM was translated to in vivo leukocyte recruitment as COAM binding to mouse GCP-2 promoted a fast and cooperative neutrophil chemotaxis into the peritoneal cavity of mice. These results showed that the formation of a binding complex between COAM and specific chemokines might represent the underlying mechanism of the observed effect on leukocyte migration in vivo. Binding of COAM to GCP-2 and other chemokines might provide an endogenous chemotactic force, influencing selective and directional leukocyte chemotaxis.In conclusion, central to the mechanism of antiviral protection of the polysaccharide derivative COAM is the concept of leukocyte chemotaxis. Leukocytes, in particular neutrophils, can be tuned toward antiviral defense by this pharmacological molecule that selectively binds chemokines with high affinity. These glycosaminoglycan mimetic features establish COAM as an immunomodulator and an interesting probe with the capacity to influence chemokine gradients and selectivity of leukocyte responses in vivo. Furthermore, COAM constitutes a novel tool to investigate how cells of the myeloid lineage, in particular neutrophils, can influence the pathogenesis of viral infections and other immunopathologies. As the field of viral immunology is rapidly evolving, comprehension concerning the mode of action of antiviral immunomodulators might reveal new concepts which may stimulate future exploration of new antiviral therapeutic strategies.

Publication year:2011

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