Carol B. Martin

Expression of complement protein C5a in a murine mammary cancer model: tumor regression by interference with the cell cycle

The C5a anaphylatoxin protein plays a central role in inflammation associated with complement activation. This protein is commonly regarded as one of the most potent inducers of the inflammatory response and a C5a peptide agonist was used as a molecular adjuvant. However, the full length C5a protein has not been tested as a potential tumor therapy. In this report, we describe the creation of a mini-gene construct that directs C5a expression to any cell of interest. Functional expression could be demonstrated in the murine mammary sarcoma, EMT6. When C5a expressing cells were injected into syngeneic mice, most C5a-expressing clones had significantly reduced tumor growth. Further characterization of a clone expressing low levels of C5a demonstrated that one-third of mice injected with this line had complete tumor regression. The mice whose tumors regressed were immune to subsequent challenge with unmodified EMT6 cells, suggesting that a component of the innate immune response can be used to augment adaptive immunity. Cellular analyses demonstrated that a significant difference in actual tumor cell number could be detected as early as day 10. A block in cell cycle progression was evident at all time points and high levels of apoptosis were observed early in the regression event. These data demonstrate that the complement protein C5a can play a significant protective role in tumor immunity.

CNS-specific expression of C3a and C5a exacerbate demyelination severity in the cuprizone model

Demyelination in the central nervous system (CNS) is known to involve several immune effector mechanisms, including complement proteins. Local production of complement by glial cells in the brain can be both harmful and protective. To investigate the roles of C3a and C5a in demyelination and remyelination pathology we utilized the cuprizone model. Transgenic mice expressing C3a or C5a under the control of the glial fibrillary acidic protein (GFAP) promoter had exacerbated demyelination and slightly delayed remyelination in the corpus callosum compared to WT mice. C3a and C5a transgenic mice had increased cellularity in the corpus callosum due to increase activation and/or migration of microglia. Oligodendrocytes migrated to the corpus callosum in higher numbers during early remyelination events in C3a and C5a transgenic mice, thus enabling these mice to remyelinate as effectively as WT mice by the end of the 10 week study. To determine the effects of C3a and/or C5a on individual glial subsets, we created murine recombinant C3a and C5a proteins. When microglia and mixed glial cultures were stimulated with C3a and/or C5a, we observed an increase in the production of proinflammatory cytokines and chemokines. In contrast, astrocytes had decreased cytokine and chemokine production in the presence of C3a and/or C5a. We also found that the MAPK pathway proteins JNK and ERK1/2 were activated in glia upon stimulation with C3a and C5a. Overall, our findings show that although C3a and C5a production in the brain play a negative role during demyelination, these proteins may aid in remyelination.

Characterization of the Murine C3a Receptor Enhancer-Promoter: Expression Control by an Activator Protein 1 Sequence and an Ets-Like Site

The complement anaphylatoxins, C3a and C5a, exert their effects by binding to their respective receptors. A number of studies have implicated these proteins in human disease, yet little is known about anaphylatoxin receptor gene regulation. In this report, we demonstrate that most of the regulatory functions in the murine C3aR gene lie within 50 bp of the transcription start site. This region is critical for macrophage expression but does not have activity in a nonexpressing melanoma cell line. Within this small region are putative consensus binding sites for AP-1, NF-κB, Ets, and GATA transcription factors. Lack of a corresponding NF-κB site in the human sequence and lack of DNA binding activity in macrophage nuclear extracts suggests that the NF-κB site is nonfunctional. Luciferase data demonstrate that the GATA site functions as a negative regulatory element in RAW 264.7 macrophages. The AP-1 and Ets sites are critical for C3aR reporter gene expression, such that when each is mutated, a significant loss of activity is observed. Furthermore, we demonstrate that these sequences cooperate to mediate both basal and LPS-induced expression of C3aR. Interestingly, EMSA analyses demonstrate that the AP-1 site binds to c-Jun, and in vivo footprinting shows a typical footprint in this site, but the Ets site does not have a “typical” Ets footprint and does not bind to Ets-1/2 proteins in RAW 264.7 extracts. These data suggest that, although the control region for C3aR is small, interaction of several transcription factors can lead to complex patterns of gene regulation.

Astrocyte‐specific expression of a soluble form of the murine complement control protein Crry confers demyelination protection in the cuprizone model

Complement has been implicated as a potential effector mechanism in neurodegeneration; yet the precise role of complement in this process remains elusive. In this report, we have utilized the cuprizone model of demyelination‐remyelination to examine the contribution of complement to disease. C1q deposition was observed in the corpus callosum of C57BL/6 mice during demyelination, suggesting complement activation by apoptotic oligodendrocyte debris. Simultaneously, these mice lost expression of the rodent complement regulatory protein, Crry. A soluble CNS‐specific form of the Crry protein (sCrry) expressed in a transgenic mouse under the control of an astrocyte‐specific promoter was induced in the corpus callosum during cuprizone treatment. Expression of this protein completely protected the mice from demyelination. Interestingly, sCrry mice had low levels of demyelination at later times when control mice were remyelinating. Although the sCrry transgenic mice had lower levels of demyelination, there was no decrease in overall cellularity, however there were decreased numbers of microglia in the sCrry mice relative to controls. Strikingly, sCrry mice had early recovery of mature oligodendrocytes, although they later disappeared. TUNEL staining suggested that production of the sCrry protein in the transgenic mice protected from a late apoptosis event at 3 weeks of cuprizone treatment. Our data suggest complement provides some protection of mature oligodendrocytes during cuprizone treatment but may be critical for subsequent remyelination events. These data suggest that temporal restriction of complement inhibition may be required in some disease settings.

c-Jun and c-Fos regulate the complement factor H promoter in murine astrocytes

The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins, including complement factor H (CFH), a critical regulator of the alternative pathway of complement activation. Given its regulatory function, mutations in CFH have been implicated in diseases such as age-related macular degeneration and membranoproliferative glomerulonephritis, and central nervous system diseases such as Alzheimers disease, Parkinsons disease, and a demyelinating murine model, experimental autoimmune encephalomyelitis (EAE). There have been few investigations on the transcriptional regulation of CFH in the brain and CNS. Our studies show that CFH mRNA is present in several CNS cell types. The murine CFH (mCFH) promoter was cloned and examined through truncation constructs and we show that specific regions throughout the promoter contain enhancers and repressors that are positively regulated by inflammatory cytokines in astrocytes. Database mining of these regions indicated transcription factor binding sites conserved between different species, which led to the investigation of specific transcription factor binding interactions in a 241 base pair (bp) region at -416 bp to -175 bp that showed the strongest activity. Through supershift analysis, it was determined that c-Jun and c-Fos interact with the CFH promoter in astrocytes in this region. These results suggest a relationship between cell cycle and complement regulation, and how these transcription factors and CFH affect disease will be a valuable area of investigation.

Addition of αGal HyperAcute™ technology to recombinant avian influenza vaccines induces strong low-dose antibody responses

Highly pathogenic avian influenza represents a severe public health threat. Over the last decade, the demand for highly efficacious vaccines against avian influenza viruses has grown, especially after the 2013 H7N9 outbreak in China that resulted in over 600 human cases with over 200 deaths. Currently, there are several H5N1 and H7N9 influenza vaccines in clinical trials, all of which employ traditional oil-in-water adjuvants due to the poor immunogenicity of avian influenza virus antigens. In this study, we developed potent recombinant avian influenza vaccine candidates using HyperAcute™ Technology, which takes advantage of naturally-acquired anti-αGal immunity in humans. We successfully generated αGal-positive recombinant protein and virus-like particle vaccine candidates of H5N1 and H7N9 influenza strains using either biological or our novel CarboLink chemical αGal modification techniques. Strikingly, two doses of 100 ng αGal-modified vaccine, with no traditional adjuvant, was able to induce a much stronger humoral response in αGT BALB/c knockout mice (the only experimental system readily available for testing αGal in vivo) than unmodified vaccines even at 10-fold higher dose (1000 ng/dose). Our data strongly suggest that αGal modification significantly enhances the humoral immunogenicity of the recombinant influenza vaccine candidates. Use of αGal HyperAcute™ technology allows significant dose-sparing while retaining desired immunogenicity. Our success in the development of highly potent H5N1 and H7N9 vaccine candidates demonstrated the potential of αGal HyperAcute™ technology for the development of vaccines against other infectious diseases.