Maria R. Bassi

Matrix metalloproteinase-8 overexpression prevents proper tissue repair.

BACKGROUND The collagenolytic matrix metalloproteinase-8 (MMP-8) is essential for normal tissue repair but is often overexpressed in wounds with disrupted healing. Our aim was to study the impact of a local excess of this neutrophil-derived proteinase on wound healing using recombinant adenovirus-driven transduction of full-length Mmp8 (AdMMP-8). METHODS The effect of MMP-8 overexpression was evaluated in dermal fibroblasts and in two wound healing models in male Wistar rats: subcutaneously positioned ePTFE catheters and linear incisional skin wounds. RESULTS Fibroblasts transduced with AdMMP-8 secreted MMP-8 with type I collagenolytic activity that could be blocked by a selective MMP-8 inhibitor. AdMMP-8 (5 × 10(10) viral particles) administered in homologous fibrin increased MMP-8 mRNA (P < .05) levels compared to parallel wounds treated with a control adenovirus expressing lacZ (AdLacZ). Impaired wound healing was demonstrated with AdMMP-8 by decreased collagen deposition and breaking strength of incisional wounds on day 7 compared to AdLacZ-treated wounds (P < .05). We found no significant effect of AdMMP-8 on mRNA levels of MMP-9, COL1A1, or COL3A1, but AdMMP-8 treatment decreased the number of neutrophils. In the incisional wounds, MMP-8 gene transfer was not associated with significant changes in macrophage numbers or amount of granulation tissue but did increase MMP-8 protein by 76% (P < .01) and decrease type I collagen protein by 29% (P < .05) compared with AdLacZ. CONCLUSION These results demonstrate that superphysiologic levels of the proteinase MMP-8 can result in decreased collagen and lead to impaired wound healing. This observation makes MMP-8 a potential drug target in compromised human wound healing associated with MMP-8 overexpression.

CD8+ T Cells Complement Antibodies in Protecting against Yellow Fever Virus

The attenuated yellow fever (YF) vaccine (YF-17D) was developed in the 1930s, yet little is known about the protective mechanisms underlying its efficiency. In this study, we analyzed the relative contribution of cell-mediated and humoral immunity to the vaccine-induced protection in a murine model of YF-17D infection. Using different strains of knockout mice, we found that CD4+ T cells, B cells, and Abs are required for full clinical protection of vaccinated mice, whereas CD8+ T cells are dispensable for long-term survival after intracerebral challenge. However, by analyzing the immune response inside the infected CNS, we observed an accelerated T cell influx into the brain after intracerebral challenge of vaccinated mice, and this T cell recruitment correlated with improved virus control in the brain. Using mice deficient in B cells we found that, in the absence of Abs, YF vaccination can still induce some antiviral protection, and in vivo depletion of CD8+ T cells from these animals revealed a pivotal role for CD8+ T cells in controlling virus replication in the absence of a humoral response. Finally, we demonstrated that effector CD8+ T cells also contribute to viral control in the presence of circulating YF-specific Abs. To our knowledge, this is the first time that YF-specific CD8+ T cells have been demonstrated to possess antiviral activity in vivo.

Transgenic Rescue of Adipocyte Glucose-dependent Insulinotropic Polypeptide Receptor Expression Restores High Fat Diet-induced Body Weight Gain

Background: Glucose-dependent insulinotropic polypeptide (GIP) is pursued as an anti-obesity target. Results: The adipocyte GIP receptor (GIPr) promotes high fat diet (HFD)-induced body weight gain by an increase in lean mass rather than fat mass in mice. Conclusion: The adipocyte GIPr regulates both body weight and body composition. Significance: Targeting the GIPr may have effects beyond lipid storage and acute glucose metabolism. The glucose-dependent insulinotropic polypeptide receptor (GIPr) has been implicated in high fat diet-induced obesity and is proposed as an anti-obesity target despite an uncertainty regarding the mechanism of action. To independently investigate the contribution of the insulinotropic effects and the direct effects on adipose tissue, we generated transgenic mice with targeted expression of the human GIPr to white adipose tissue or beta-cells, respectively. These mice were then cross-bred with the GIPr knock-out strain. The central findings of the study are that mice with GIPr expression targeted to adipose tissue have a similar high fat diet -induced body weight gain as control mice, significantly greater than the weight gain in mice with a general ablation of the receptor. Surprisingly, this difference was due to an increase in total lean body mass rather than a gain in total fat mass that was similar between the groups. In contrast, glucose-dependent insulinotropic polypeptide-mediated insulin secretion does not seem to be important for regulation of body weight after high fat feeding. The study supports a role of the adipocyte GIPr in nutrient-dependent regulation of body weight and lean mass, but it does not support a direct and independent role for the adipocyte or beta-cell GIPr in promoting adipogenesis.

Pre-Existing Vector Immunity Does Not Prevent Replication Deficient Adenovirus from Inducing Efficient CD8 T-Cell Memory and Recall Responses

Adenoviral vectors have shown a great potential for vaccine development due to their inherent ability to induce potent and protective CD8 T-cell responses. However, a critical issue regarding the use of these vectors is the existence of inhibitory immunity against the most commonly used Ad5 vector in a large part of the human population. We have recently developed an improved adenoviral vaccine vector system in which the vector expresses the transgene tethered to the MHC class II associated invariant chain (Ii). To further evaluate the potential of this system, the concept of pre-existing inhibitory immunity to adenoviral vectors was revisited to investigate whether the inhibition previously seen with the Ad5 vector also applied to the optimized vector system. We found this to be the case, and antibodies dominated as the mechanism underlying inhibitory vector immunity. However, presence of CD8 T cells directed against epitopes in the adenoviral vector seemed to correlate with repression of the induced response in re-vaccinated B-cell deficient mice. More importantly, despite a repressed primary effector CD8 T-cell response in Ad5-immune animals subjected to vaccination, memory T cells were generated that provided the foundation for an efficient recall response and protection upon subsequent viral challenge. Furthermore, the transgene specific response could be efficiently boosted by homologous re-immunization. Taken together, these studies indicate that adenoviral vectors can be used to induce efficient CD8 T-cell memory even in individuals with pre-existing vector immunity.

Vaccination with Replication Deficient Adenovectors Encoding YF-17D Antigens Induces Long-Lasting Protection from Severe Yellow Fever Virus Infection in Mice

The live attenuated yellow fever vaccine (YF-17D) has been successfully used for more than 70 years. It is generally considered a safe vaccine, however, recent reports of serious adverse events following vaccination have raised concerns and led to suggestions that even safer YF vaccines should be developed. Replication deficient adenoviruses (Ad) have been widely evaluated as recombinant vectors, particularly in the context of prophylactic vaccination against viral infections in which induction of CD8+ T-cell mediated immunity is crucial, but potent antibody responses may also be elicited using these vectors. In this study, we present two adenobased vectors targeting non-structural and structural YF antigens and characterize their immunological properties. We report that a single immunization with an Ad-vector encoding the non-structural protein 3 from YF-17D could elicit a strong CD8+ T-cell response, which afforded a high degree of protection from subsequent intracranial challenge of vaccinated mice. However, full protection was only observed using a vector encoding the structural proteins from YF-17D. This vector elicited virus-specific CD8+ T cells as well as neutralizing antibodies, and both components were shown to be important for protection thus mimicking the situation recently uncovered in YF-17D vaccinated mice. Considering that Ad-vectors are very safe, easy to produce and highly immunogenic in humans, our data indicate that a replication deficient adenovector-based YF vaccine may represent a safe and efficient alternative to the classical live attenuated YF vaccine and should be further tested.

Suppressors of Cytokine Signaling 1 and 3 Are Upregulated in Brain Resident Cells in Response to Virus-Induced Inflammation of the Central Nervous System via at Least Two Distinctive Pathways

ABSTRACT Suppressors of cytokine signaling (SOCS) proteins are intracellular proteins that inhibit cytokine signaling in a variety of cell types. A number of viral infections have been associated with SOCS upregulation; however, not much is known about the mechanisms regulating SOCS expression during viral infection. In this study, we used two pathologically distinct intracerebral (i.c.) infection models to characterize temporal and spatial aspects of SOCS expression in the virus-infected central nervous system (CNS), and by employing various knockout mouse models, we sought to identify regulatory mechanisms that may underlie a virus induced upregulation of SOCS in the CNS. We found that i.c. infection with either lymphocytic choriomeningitis virus (LCMV) or yellow fever virus (YF) results in gradual upregulation of SOCS1/3 mRNA expression peaking at day 7 postinfection (p.i.). In the LCMV model, SOCS mRNA was expressed in brain resident cells, including astrocytes and some neurons, and for SOCS1 in particular this upregulation was almost entirely mediated by gamma interferon (IFN-γ) produced by infiltrating T cells. After infection with YF, we also found SOCS expression to be upregulated in brain resident cells with a peak on day 7 p.i., but in this model, the upregulation was only partially dependent on IFN-γ and T cells, indicating that at least one other mediator was involved in the upregulation of SOCS following YF infection. We conclude that virus-induced inflammation of the CNS is associated with upregulation of SOCS1/3 mRNA expression in brain resident cells and that at least two distinctive pathways can lead to this upregulation. IMPORTANCE In the present report, we have studied the induction of SOCS1 and SOCS3 expression in the context of virus-induced CNS infection. We found that both a noncytolytic and a cytolytic virus induce marked upregulation of SOCS1 and -3 expression. Notably, the kinetics of the observed upregulation follows that of activity within proinflammatory signaling pathways and, interestingly, type II interferon (IFN), which is also a key inducer of inflammatory mediators, seems to be essential in initiating this counterinflammatory response. Another key observation is that not only cells of the immune system but also CNS resident cells are actively involved in both the pro- and the counterinflammatory immune circuits; thus, for example, astrocytes upregulate both C-X-C-motif chemokine 10 (CXCL10) and SOCS when exposed to type II IFN in vivo.

Adaptive immune responses to booster vaccination against yellow fever virus are much reduced compared to those after primary vaccination

Outbreaks of Yellow Fever occur regularly in endemic areas of Africa and South America frequently leading to mass vaccination campaigns straining the availability of the attenuated Yellow Fever vaccine, YF-17D. The WHO has recently decided to discontinue regular booster-vaccinations since a single vaccination is deemed to confer life-long immune protection. Here, we have examined humoral (neutralizing antibody) and cellular (CD8 and CD4 T cell) immune responses in primary and booster vaccinees (the latter spanning 8 to 36 years after primary vaccination). After primary vaccination, we observed strong cellular immune responses with T cell activation peaking ≈2 weeks and subsiding to background levels ≈ 4 weeks post-vaccination. The number of antigen-specific CD8+ T cells declined over the following years. In >90% of vaccinees, in vitro expandable T cells could still be detected >10 years post-vaccination. Although most vaccinees responded to a booster vaccination, both the humoral and cellular immune responses observed following booster vaccination were strikingly reduced compared to primary responses. This suggests that pre-existing immunity efficiently controls booster inoculums of YF-17D. In a situation with epidemic outbreaks, one could argue that a more efficient use of a limited supply of the vaccine would be to focus on primary vaccinations.

EBI2 overexpression in mice leads to B1 B cell expansion and chronic lymphocytic leukemia-(CLL)-like B cell malignancies.

Human and mouse chronic lymphocytic leukemia (CLL) develops from CD5+ B cells that in mice and macaques are known to define the distinct B1a B-cell lineage. B1a cells are characterized by lack of germinal center (GC) development, and the B1a cell population is increased in mice with reduced GC formation. As a major mediator of follicular B-cell migration, the G protein-coupled receptor Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) directs B-cell migration in the lymphoid follicles in response to its endogenous ligands, oxysterols. Thus, upregulation of EBI2 drives the B cells toward the extrafollicular area, whereas downregulation is essential for GC formation. We therefore speculated whether increased expression of EBI2 would lead to an expanded B1 cell subset and, ultimately, progression to CLL. Here, we demonstrate that B-cell-targeted expression of human EBI2 (hEBI2) in mice reduces GC-dependent immune responses, reduces total immunoglobulin M (IgM) and IgG levels, and leads to increased proliferation and upregulation of cellular oncogenes. Furthermore, hEBI2 overexpression leads to an abnormally expanded CD5+ B1a B-cell subset (present as early as 4 days after birth), late-onset lymphoid cancer development, and premature death. These findings are highly similar to those observed in CLL patients and identify EBI2 as a promoter of B-cell malignancies.

Early life vaccination: Generation of adult-quality memory CD8+ T cells in infant mice using non-replicating adenoviral vectors

Intracellular pathogens represent a serious threat during early life. Importantly, even though the immune system of newborns may be characterized as developmentally immature, with a propensity to develop Th2 immunity, significant CD8+ T-cell responses may still be elicited in the context of optimal priming. Replication deficient adenoviral vectors have been demonstrated to induce potent CD8+ T-cell response in mice, primates and humans. The aim of the present study was therefore to assess whether replication-deficient adenovectors could overcome the risk of overwhelming antigen stimulation during the first period of life and provide a pertinent alternative in infant vaccinology. To address this, infant mice were vaccinated with three different adenoviral vectors and the CD8+ T-cell response after early life vaccination was explored. We assessed the frequency, polyfunctionality and in vivo cytotoxicity of the elicited memory CD8+ T cells, as well as the potential of these cells to respond to secondary infections and confer protection. We further tested the impact of maternal immunity against our replication-deficient adenoviral vector during early life vaccination. Overall, our results indicate that memory CD8+ T cells induced by adenoviral vectors in infant mice are of good quality and match those elicited in the adult host.