Julia D. Rempel

Differential regulation of innate and adaptive immune responses in viral encephalitis

Abstract Viral encephalitis is a global health concern. The ability of a virus to modulate the immune response can have a pivotal effect on the course of disease and the fate of the infected host. In this study, we sought to understand the immunological basis for the fatal encephalitis following infection with the murine coronavirus, mouse hepatitis virus (MHV)-JHM, in contrast with the more attenuated MHV-A59. Distinct glial cell cytokine and chemokine response patterns were observed within 3 days after infection, became progressively more polarized during the course of infection and with the infiltration of leukocytes. In the brain, MHV-JHM infection induced strong accumulation of IFNβ mRNA relative to IFNγ mRNA. This trend was reversed in MHV-A59 infection and was accompanied by increased CD8 T cell infiltration into brain compared to MHV-JHM infection. Increased apoptosis appeared to contribute to the diminished presence of CD8 T cells in MHV-JHM-infected brain with the consequence of a lower potential for IFNγ production and antiviral activity. MHV-JHM infection also induced sustained mRNA accumulation of the innate immune response products interleukin (IL)-6 and IL-1. Furthermore, high levels of macrophage-inflammatory protein (MIP)-1α, MIP-1β, and MIP-2 mRNA were observed at the onset of MHV-JHM infection and correlated with a marked elevation in the number of macrophages in the brain on day 7 compared to MHV-A59 infection. These observations indicate that differences in the severity of viral encephalitis may reflect the differential ability of viruses to stimulate innate immune responses within the CNS and subsequently the character of infiltrating leukocyte populations.

Impact of aboriginal ethnicity on HCV core-induced IL-10 synthesis: Interaction with IL-10 gene polymorphisms

The host immune response is a critical determinant in viral infection outcome. Epidemiological studies indicate that North American indigenous peoples are more resistant to chronic HCV infection than other populations. Due to the prominence of IL‐10 in chronic HCV infection, we investigated the genetic tendency to produce IL‐10 in Caucasian (CA) and First Nation (FN) populations. Peripheral blood mononuclear cells (PBMCs) from CA subjects had a greater tendency to produce IL‐10 defined by allelic polymorphisms, as well as genotypes and haplotypes, at the ‐1082, ‐819, and ‐592 positions of the IL‐10 promoter. More importantly, we directly evaluated the influence of ethnicity on the ability of HCV core protein to induce IL‐10 synthesis and found significantly higher IL‐10 production by PBMCs isolated from healthy CA subjects compared with FN subjects. Further examination of the underlying relationship between core‐induced IL‐10 with the high, intermediate, and low phenotypes at the ‐1082, ‐819, and ‐592 position revealed that spontaneous and core‐induced IL‐10 synthesis tended to interact negatively with defined polymorphisms. This was particularly evident for the FN cohort, in which the relationship was strengthened by a stronger interaction of core with the low–IL‐10–producing phenotypes. As with previous studies, concanavalin A induced IL‐10 synthesis from the CA cohort positively associated with defined genetic phenotypes. Conclusion: Cells from FN subjects had a reduced capacity to produce IL‐10 in response to HCV core protein, suggesting that reduced susceptibility of FN immunity to virally induced IL‐10 synthesis might contribute to epidemiological observations of enhanced HCV clearance. (HEPATOLOGY 2007;45:623–630.)

Mouse hepatitis virus neurovirulence: evidence of a linkage between S glycoprotein expression and immunopathology

Abstract Differences in disease outcome between the highly neurovirulent MHV-JHM and mildly neurovirulent MHV-A59 have been attributed to variations within the spike (S) glycoprotein. Previously, we found that MHV-JHM neurovirulence was marked by diminished expression of interferon-γ (IFN-γ) mRNA and a reduced presence of CD8 T cells in the CNS concomitant with heightened macrophage inflammatory protein (MIP)-1 transcript levels and greater macrophage infiltration relative to MHV-A59 infection. Here, the ability of the S and non-spike genes to regulate these immune responses was evaluated using chimeric viruses. Chimeric viruses WTR13 and S4R22 were made on MHV-A59 variant backgrounds and, respectively, contained the S gene of MHV-A59 and MHV-JHM. Unexpectedly, genes other than S appeared to modulate events critical to viral replication and survival. Unlike unresolving MHV-JHM infections, the clearance of WTR13 and S4R22 infections coincided with strong IFN-γ transcription and an increase in the number of CD8 T cells infiltrating into the CNS. However, despite the absence of detectable viral titers, approximately 40% of S4R22-infected mice succumbed within 3 weeks, indicating that the enhanced mortality following S4R22 infection was not associated with high viral titers. Instead, similar to the MHV-JHM infection, reduced survival following S4R22 infection was observed in the presence of elevated MIP-1α and MIP-1β mRNA accumulation and enhanced macrophage numbers within infected brains. These observations suggest that the S protein of MHV-JHM influences neurovirulence through the induction of MIP-1α- and MIP-1β-driven macrophage immunopathology.

Expression of Fc Fragment Receptors of Immunoglobulin G (FcγRs) in Rat Hepatic Stellate Cells

Hepatic stellate cells (HSCs) are now considered the major cell type in the liver mediating the development of liver fibrosis. Recently it was demonstrated that HSCs express membrane proteins involved in antigen presentation. We further evaluate immunological properties of HSCs by examining the expression and function of the Fc fragment of immunoglobulin G (IgG) in HSCs. In this study, we document the presence of mRNAs for three FcγRs in HSCs. Ligand binding assay indicated the existence of FcγRs with different binding affinities on membranes of HSCs. We also documented that the abundance of the three Fcγ R mRNAs increased upon activation of HSCs in vitro. Moreover, an examination of the biological activities of IgG revealed that exposure to IgG significantly stimulated HSC differentiation and proliferation. Furthermore, we studied the intracellular signaling protein, LcK, in HSCs and regulation of Lck expression and phosphorylation by IgG. Although IgG did not regulate Lck abundance and phosphorylation in HSCs, highly phosphorylated Lck was present in these cells. In conclusion, we provided evidence that HSCs expresses receptors for the Fc fragment of IgG, and IgG regulates HSC differentiation and proliferation. Therefore, immunoglobulin G may play a role in HSC activation.

Endogenous IL-12 synthesis is not required to prevent hyperexpression of type 2 cytokine and antibody responses.

Endogenous IL‐12 production is hypothesized to play an essential role preventing spontaneous expression of type 2 responses, acting as a natural inhibitor limiting development of immediate hypersensitivity. Here, IL‐12‐deficient p35– / – and p40– / – mice were used to examine the role of endogenous IL‐12 and p40 homodimer during in vivo development of exogenous antigen‐driven responses. In the absence of deliberate immunization, IL‐12‐deficient mice exhibited greatly reduced serum IgG2a but IgG1 / IgE levels no higher than controls. Immunization to elicit polarized ovalbumin‐specific type 1 or type 2 dominant responses, or using Trichinella spiralis extract in the absence of adjuvants, led to IFN‐γ production of approximately 10 % of C57BL / 6 controls yet the kinetics and intensity of primary and secondary type 2 cytokine (IL‐4, IL‐5, IL‐13) and antibody (IgG1, IgE) responses, as well as functional IL‐12 receptor expression, were consistently unaltered. Thus, while IL‐12 provides an important positive signal for Th1 development, antigen exposure in its absence does not lead to generalized enhancement of type 2 cytokine or antibody responses. The data argue that endogenous IL‐12 production is not required as a constitutive negative regulator limiting induction or expression of type 2 effector responses.

Persistent pro‐inflammatory cytokines following the initiation of pegylated IFN therapy in hepatitis C infection is associated with treatment‐induced depression

Summary.  Pegylated interferon (IFN), the basis for chronic hepatitis C virus (HCV) treatment, causes depression in 30–40% of patients. The potential for cytokine mRNA patterns from baseline into early treatment to associate with the onset of treatment‐induced depression (TID) was examined. Depression was measured by the Beck Depression Inventory at baseline and weeks 2, 4, 8 and 12 of treatment (n = 38). At baseline and weeks 2 and 4, peripheral blood mononuclear cell (PMBC, n = 28), isolated ex vivo, were examined for tumour neurosis factor (TNF)‐alpha, interleukin (IL)‐1beta and IL‐10 mRNA expression. In patients that developed treatment‐induced depression, pro‐inflammatory TNF‐alpha mRNA levels from baseline into week 4 of therapy remained constant (1.1‐fold increase); whereas IL‐1beta transcripts decreased 3.5 fold. However, corresponding TNF‐alpha (3‐fold, P < 0.05) and IL‐1beta (7.5‐fold) transcript expression diminished to a greater extent in the absence of TID. Changes in TNF‐alpha mRNA values correlated to the average change in BDI scores over the 12 weeks (r = 0.56, P < 0.05). Concomitantly, anti‐inflammatory IL‐10 transcript levels decreased in (TID), relative to increased expression in the absence of TID (P < 0.05). The potential influence of IL‐10 was observed upon calculation of individual pro‐ verses anti‐inflammatory mRNA ratios. Stable in the presence of depression, TNF‐alpha/IL‐10 and IL‐1beta/IL‐10 mRNA ratios declined significantly over time in its absence (P < 0.05). This study suggests that in chronic HCV infection, upon pegylated IFN administration persistent pro‐inflammatory cytokine MRNA expression associates with TID. In contrast, therapeutic activation of mechanisms that decrease pro‐inflammatory immunity may protect against depression during therapy.

The potential influence of KIR cluster profiles on disease patterns of Canadian Aboriginals and other indigenous peoples of the Americas

Genetic differences in immune regulators influence disease resistance and susceptibility patterns. There are major health discrepancies in immune-mediated diseases between Caucasians and Canadian Aboriginal people, as well as with other indigenous people of the Americas. Environmental factors offer a limited explanation as Aboriginal people also demonstrate a rare resistance to chronic hepatitis C virus infection. Killer immunoglobulin-like receptors (KIRs) are known modulators of viral responses and autoimmune diseases. The possibility that variation in KIR cluster profiles contribute to the health outcomes of Aboriginal people was evaluated with Canadian Caucasian (n=93, population controls) and Aboriginal (n=86) individuals. Relative to Caucasians, the Aboriginal KIR cluster displayed a greater immune activating phenotype associated with genes of the B haplotype situated within the telomeric region. In conjunction, there was a decrease in the genes of the B haplotype from the centromeric region. Caucasian and Aboriginal cohorts further demonstrated distinct genotype and haplotype relationships enforcing the disconnect between the B haplotype centromeric and telomeric regions within the Aboriginal population. Moreover, Caucasian KIR cluster patterns reflected studies of Caucasians globally, as well as Asians. In contrast, the unique pattern of the Canadian Aboriginal cohort mirrored the phenotype of other indigenous peoples of the Americas, but not that of Caucasians or Asians. Taken together, these data suggest that historically indigenous peoples of the Americas were subject to immune selection processes that could be influencing the current disease resistance and susceptibility patterns of their descendents.

Antigen-specific versus total immunoglobulin synthesis: total IgE and IgG1, but not IgG2a levels predict murine antigen-specific responses.

Background: Induction of an effective antibody (Ab) response requires delivery of multiple signals to B cells. Cross-linking of the B cell antigen receptor (BCR), signaling through CD40 and CD80/86 and cytokine signals combine to induce class switching and expression of specific isotypes. These signals are principally derived from activated, antigen (Ag)-specific T cells. In contrast, IFNγ, the only cytokine known to induce class switch to IgG2a, can be produced systemically by activated NK or NKT cells, suggesting that Ag-nonspecific signals may also regulate IgG2a production. Methods: Given the potential differences in regulation between IgE/IgG1 versus IgG2a, we immunized mice on day 0 with ovalbumin (OVA) in the presence of strong type-1- or type-2-immunity-inducing adjuvants and boosted mice 4 weeks later. Mice were bled during the primary immune response and after boost to assess primary and recall Ab responses. Results: Regardless of strain of mice used, phenotype (type 1 versus type 2 dominated) or nature of the immune response induced (primary versus recall), strong correlations between OVA-specific and total IgE and IgG1 were demonstrated. In contrast, a consistent lack of correlation between OVA-specific and total IgG2a levels was observed in all but BALB/c mice. Conclusion: These data indicate that the increase in total levels of IgE/IgG1 isotypes is primarily a result of increased levels of OVA-specific Ab. In contrast, the lack of correlation between total and OVA-specific IgG2a suggests broader activation of IgG2a-producing B cells routinely occurs following exogenous Ag immunization.

Prevention of allergen-specific, Th2-biased immune responses in vivo: role of increased IL-12 and IL-18 responsiveness.

The factors that control development of adaptive responses to exogenous Ag remain incompletely understood. An ability to selectively direct immunity toward a specific phenotype would be of clinical benefit in numerous immunological disorders. Administration of chemically modified allergen glutaraldehyde-polymerized OVA (OA-POL) leads to >90% reductions in murine IgE and >500-fold increases in IgG2c responses that develop upon subsequent immunization with native Ag. In the present study, we examine the mechanisms underlying this reorientation of the type 2 dominant response that would normally develop. Lack of endogenous IL-12 or IFN-γ results in markedly reduced induction of IgG2c responses following OA-POL treatment, but only IFN-γ−/− mice demonstrate reduced capacity to prevent IgE induction. This indicates that while both IL-12 and IFN-γ are critical promoters of type 1 immunity, only IFN-γ is required to maximally inhibit development of type 2 immune responses. Compared with OVA-immunized mice, CD69+ T cells from OA-POL-immunized mice demonstrate elevated IL-12Rβ2, IL-18Rα, and IL-18Rβ mRNA levels, as well as increased IFN-γ production in response to rIL-12 or rIL-18 stimulation. Collectively, these data indicate that preventing induction of type 2 immune responses is critically dependent on altered T cell responsiveness to these cytokines. The finding that targeted, Ag-specific manipulation of IL-12 and IL-18 responsiveness can be used to shape the phenotype of the dominant immune response that develops suggests that specifically targeting IL-12 and IL-18 receptor expression may offer clinical options for clinical prophylaxis or intervention.

Preliminary analysis of immune activation in early onset type 2 diabetes.

Introduction First Nations and other Aboriginal children are disproportionately affected by cardiometabolic diseases, including type 2 diabetes (T2D). In T2D, the disruption of insulin signalling can be driven by pro-inflammatory immunity. Pro-inflammatory responses can be fueled by toll-like receptors (TLR) on immune cells such as peripheral blood mononuclear cells (PBMC, a white blood cell population). TLR4 can bind to lipids from bacteria and food sources activating PBMC to produce cytokines tumour necrosis factor (TNF)-α and interleukin (IL)-1β. These cytokines can interfere with insulin signalling. Here, we seek to understand how TLR4 activation may be involved in early onset T2D. We hypothesized that immune cells from youth with T2D (n=8) would be more reactive upon TLR4 stimulation relative to cells from age and body mass index (BMI)-matched controls without T2D (n=8). Methods Serum samples were assayed for adipokines (adiponectin and leptin), as well as cytokines. Freshly isolated PBMC were examined for immune reactivity upon culture with TLR4 ligands bacterial lipopolysaccharide (LPS, 2 and 0.2 ng/ml) and the fatty acid palmitate (200 µM). Culture supernatants were evaluated for the amount of TNF-α and IL-1β produced by PBMC. Results Youth with T2D displayed lower median serum adiponectin levels compared to controls (395 vs. 904 ng/ml, p<0.05). PBMC isolated from youth with and without T2D produced similar levels of TNF-α and IL-1β after exposure to the higher LPS concentration. However, at the low LPS dose the T2D cohort exhibited enhanced IL-1β synthesis relative to the control cohort. Additionally, exposure to palmitate resulted in greater IL-1β synthesis in PBMCs isolated from youth with T2D versus controls (p<0.05). These differences in cytokine production corresponded to greater monocyte activation in the T2D cohort. Conclusion These preliminary results suggest that cellular immune responses are exaggerated in T2D, particularly with respect to IL-1β activity. These studies aim to improve the understanding of the biology behind early onset T2D and its vascular complications that burden First Nations people.