Olivia J. White

Ovalbumin-sensitized mice are good models for airway hyperresponsiveness but not acute physiological responses to allergen inhalation

Background Asthma is a chronic inflammatory disease that is characterized clinically by airway hyperresponsiveness (AHR) to bronchoconstricting agents. The physiological response of the asthmatic lung to inhaled allergen is often characterized by two distinct phases: an early‐phase response (EPR) within the first hour following exposure that subsides and a late‐phase response (LPR) that is more prolonged and may occur several hours later. Mouse models of asthma have become increasingly popular and should be designed to exhibit an EPR, LPR and AHR.

Innate IFNs and Plasmacytoid Dendritic Cells Constrain Th2 Cytokine Responses to Rhinovirus: A Regulatory Mechanism with Relevance to Asthma

Human rhinoviruses (RV) cause only minor illness in healthy individuals, but can have deleterious consequences in people with asthma. This study sought to examine normal homeostatic mechanisms regulating adaptive immunity to RV in healthy humans, focusing on effects of IFN-αβ and plasmacytoid dendritic cells (pDC) on Th2 immune responses. PBMC were isolated from 27 healthy individuals and cultured with RV16 for up to 5 d. In some experiments, IFN-αβ was neutralized using a decoy receptor that blocks IFN signaling, whereas specific dendritic cell subsets were depleted from cultures with immune-magnetic beads. RV16 induced robust expression of IFN-α, IFN-β, multiple IFN-stimulated genes, and T cell-polarizing factors within the first 24 h. At 5 d, the production of memory T cell-derived IFN-γ, IL-10, and IL-13, but not IL-17A, was significantly elevated. Neutralizing the effects of type-I IFN with the decoy receptor B18R led to a significant increase in IL-13 synthesis, but had no effect on IFN-γ synthesis. Depletion of pDC from RV-stimulated cultures markedly inhibited IFN-α secretion, and led to a significant increase in expression and production of the Th2 cytokines IL-5 (p = 0.02), IL-9 (p < 0.01), and IL-13 (p < 0.01), but had no effect on IFN-γ synthesis. Depletion of CD1c+ dendritic cells did not alter cytokine synthesis. In healthy humans, pDC and the IFN-αβ they secrete selectively constrain Th2 cytokine synthesis following RV exposure in vitro. This important regulatory mechanism may be lost in asthma; deficient IFN-αβ synthesis and/or pDC dysfunction have the potential to contribute to asthma exacerbations during RV infections.

Th2-polarisation of cellular immune memory to neonatal pertussis vaccination.

Current infant vaccination against pertussis in North America and Australia requires three doses of vaccines including diphtheria, tetanus and acellular pertussis antigens (DTaP) at 2, 4 and 6 months of age. Interest is growing in the possibility that vaccination at birth might provide earlier protection of infants, but early vaccination also gives rise to concerns over the potential for excessive Th2-polarisation of pertussis-specific T-cell memory profiles. We evaluated this issue as part of a small pilot study comparing infants receiving a monovalent acellular pertussis vaccine (aP) at birth or birth and at 1 month, followed by DTaP at 2, 4 and 6 months with infants receiving DTaP only from 2 months. We compared in vitro Th-memory responses at 8 months and pertussis-specific IgG in serum at 2, 4, 6 and 8 months. Neonatal vaccination elicited earlier IgG responses, but accompanying Th-memory profiles displayed a strong Th2 bias with high IL-5 and IL-13 production. The correlation between T-cell memory profiles and other clinical outcomes should be evaluated in larger trials of neonatal aP vaccine.

Tolerance or Immunity to a Tumor Antigen Expressed in Somatic Cells Can Be Determined by Systemic Proinflammatory Signals at the Time of First Antigen Exposure

Mice transgenic for the E7 tumor Ag of human papillomavirus type 16, driven from a keratin 14 promoter, express E7 in keratinocytes but not dendritic cells. Grafted E7-transgenic skin is not rejected by E7-immunized mice that reject E7-transduced transplantable tumors. Rejection of recently transplanted E7-transgenic skin grafts, but not of control nontransgenic grafts or of established E7-transgenic grafts, is induced by systemic administration of live or killed Listeria monocytogenes or of endotoxin. Graft recipients that reject an E7 graft reject a subsequent E7 graft more rapidly and without further L. monocytogenes exposure, whereas recipients of an E7 graft given without L. monocytogenes do not reject a second graft, even if given with L. monocytogenes. Thus, cross-presentation of E7 from keratinocytes to the adaptive immune system occurs with or without a proinflammatory stimulus, but proinflammatory stimuli at the time of first cross-presentation of Ag can determine the nature of the immune response to the Ag. Furthermore, immune effector mechanisms responsible for rejection of epithelium expressing a tumor Ag in keratinocytes are different from those that reject an E7-expressing transplantable tumor. These observations have implications for immunotherapy for epithelial cancers.

Asthma is associated with multiple alterations in anti-viral innate signalling pathways.

Background Human rhinovirus (HRV) infection is a major trigger for asthma exacerbations. Anti-viral immunity appears to be abnormal in asthma, with immune dysfunction reported in both airway structural cells and migratory, bone marrow derived cells. Though decreased capacity to produce anti-viral interferons (IFNs) has been reported in asthma, a detailed analysis of the molecular events involved has not been undertaken. Objective To compare the molecular pathway controlling type I IFN synthesis in HRV-stimulated peripheral blood mononuclear cells (PBMC) from asthmatic and healthy subjects. Methods PBMC from 22 allergic asthmatics and 20 healthy donors were cultured with HRV for 24 hours. Multiple components of the Toll-like receptor (TLR), IFN regulatory and NFκβ pathways were compared at the mRNA and protein level. Results Multiple deficiencies in the innate immune response to HRV were identified in asthma, with significantly lower expression of IFNα, IFNβ and interferon stimulated genes than in healthy subjects. This was accompanied by reduced expression of intra-cellular signalling molecules including interferon regulatory factors (IRF1, IRF7), NF-κB family members (p50, p52, p65 and IκKα) and STAT1, and by reduced responsiveness to TLR7/TLR8 activation. These observations could not be attributed to alterations in the numbers of dendritic cell (DC) subsets in asthma or baseline expression of the viral RNA sensing receptors TLR7/TLR8. In healthy subjects, blocking the activity of type-I IFN or depleting plasmacytoid DC recapitulated many of the abnormalities observed in asthma. Conclusions Multiple abnormalities in innate anti-viral signalling pathways were identified in asthma, with deficiencies in both IFN-dependent and IFN-independent molecules identified.

Airway hyperresponsiveness is associated with activated CD4+ T cells in the airways

It is widely accepted that atopic asthma depends on an allergic response in the airway, yet the immune mechanisms that underlie the development of airway hyperresponsiveness (AHR) are poorly understood. Mouse models of asthma have been developed to study the pathobiology of this disease, but there is considerable strain variation in the induction of allergic disease and AHR. The aim of this study was to compare the development of AHR in BALB/c, 129/Sv, and C57BL/6 mice after sensitization and challenge with ovalbumin (OVA). AHR to methacholine was measured using a modification of the forced oscillation technique in anesthetized, tracheostomized mice to distinguish between airway and parenchymal responses. Whereas all strains showed signs of allergic sensitization, BALB/c was the only strain to develop AHR, which was associated with the highest number of activated (CD69(+)) CD4(+) T cells in the airway wall and the highest levels of circulating OVA-specific IgG(1). AHR did not correlate with total or antigen-specific IgE. We assessed the relative contribution of CD4(+) T cells and specific IgG(1) to the development of AHR in BALB/c mice using adoptive transfer of OVA-specific CD4(+) T cells from DO11.10 mice. AHR developed in these mice in a progressive fashion following multiple OVA challenges. There was no evidence that antigen-specific antibody had a synergistic effect in this model, and we concluded that the number of antigen-specific T cells activated and recruited to the airway wall was crucial for development of AHR.

A genomics-based approach to assessment of vaccine safety and immunogenicity in children

Immune responses to vaccines in infants and young children are typically Th2-biased, giving rise to concerns regarding potential atopy-like side effects, and antagonism of Th1-associated sterilising immunity. Conventional immunological methodology has limited capacity to effectively address these problems because of the inherent complexity of the immune responses involved. In the present study, we sought to develop an unbiased systems biology approach to elucidate superficially similar Th2-associated responses to paediatric vaccines and allergens, and to differentiate between them via gene coexpression network analysis. We demonstrate below that in immune responses to the diptheria/acellular pertussis/tetanus and pneumococcal polysaccharide conjugate vaccines, potentially antagonistic Th1-/IFN-associated and Th2-associated gene networks coexist in an apparent state of dynamic equilibrium, whereas in Th2-dominant allergen-specific responses of atopics the Th1 and IFN networks are respectively disrupted and downregulated. Capacity to detect and interpret these covert differences between responses to vaccines and allergens relies on the use of sophisticated algorithms that underpin coexpression network analysis, which identify genes that function co-ordinately in complex pathways. This methodology has significant potential to identify covert interactions between inflammatory pathways triggered by vaccination, and as such may be a useful tool in prediction of vaccine safety/efficacy.

Innate interferons inhibit allergen and microbial specific T H 2 responses

Several studies provided evidence of innate interferons (IFNs) regulating TH2 cytokine production using purified CD4+ memory cells and TH2 polarisation via interleukin‐4 (IL‐4). Vitally, none of these previous studies examined IFN attenuation of TH2 responses to allergen or antigen. This study therefore sought to investigate the abrogation of specific allergen‐ and antigen‐stimulated TH2 response in peripheral blood mononuclear cells (PBMC) derived from 12 sensitised individuals by IFN‐β and IFN‐λ. PBMC were cultured in the presence of house dust mite (HDM) allergen, rhinovirus (RV), influenza vaccine and tetanus toxoid (TT)±either IFN‐β or IFN‐λ for 3 and 5 days. IFN‐γ, IL‐5 and IL‐13 protein levels were measured by ELISA. Quantitative PCR (qPCR) was used to investigate induction of genes involved in control of TH2 cytokines. No alteration in TH1 IFN‐γ allergen/antigen response was observed with addition of IFN‐β or IFN‐λ. Consistent abrogation of TH2 response to HDM and influenza was observed with IFN‐β at both time points; attenuation was observed by day 5 with RV and TT. IFN‐λ had no consistent effect on TH2 production except in the presence of RV (multiplicity of infection=5); a decrease in IL‐5 alone was observed in the presence of trivalent inactivated influenza vaccine. GATA binding protein 3 (GATA3) and suppressors of cytokine signalling3 mRNA were differentially regulated in HDM and influenza‐stimulated cultures±IFN‐β. We concluded that IFN‐β produced a strong and consistent abrogation of TH2 cytokine production in the presence of a range of allergen and antigen stimulants.

Impact of sex steroid ablation on viral, tumour and vaccine responses in aged mice.

Recent evidence suggests that the decline in resistance to viral infections with age occurs predominantly as a result of a gradual loss of naïve antigen-specific T cells. As such, restoration of the naïve T cell repertoire to levels seen in young healthy adults may improve defence against infection in the aged. We have previously shown that sex steroid ablation (SSA) rejuvenates the ageing thymus and increases thymic export of naïve T cells, but it remains unclear whether T cell responses are improved. Using mouse models of clinically relevant diseases, we now demonstrate that SSA increases the number of naïve T cells able to respond to antigen, thereby enhancing effector responses in aged mice. Specifically, aged mice exhibit a delay in clearing influenza A virus, which correlates with diminished specific cytotoxic activity. This is due to a decreased magnitude of response and not an intrinsic defect in effector T cell function. Upon SSA, aged mice exhibit increased T cell responsiveness that restores efficient viral clearance. We further demonstrate that SSA decreases the incidence of an inducible tumour in aged mice and can potentially increase their responsiveness to a low-dose human papillomavirus vaccine in clearing pre-formed tumours. As thymectomy abrogates the increase in T cell numbers and responsiveness following SSA, we propose that the T cell effects of SSA are dependent on thymic reactivation and subsequent replenishment of the peripheral T cell pool with newly emigrated naïve T cells. These findings have important implications for strategies to improve protection from infection and responsiveness to vaccination in the aged.

Antibody and cell-mediated immunity to pertussis 4 years after monovalent acellular pertussis vaccine at birth.

Background: In a previous study, we found that monovalent acellular pertussis (aP) vaccine at birth and 1 month achieves higher IgG antibody (Ab) levels to pertussis toxoid (PT), filamentous hemagglutinin (FHA) and pertactin by 8 weeks, when compared with controls. Here, we report antibody and cell-mediated immune responses to 4 years of age. Methods: IgG Ab to PT, filamentous hemagglutinin and pertactin, diphtheria (D) and tetanus (T) was measured in the 3 groups (aP vaccine at birth and 1 month, aP birth only and no aP) at 2 years of age and before and after DTaP-inactivated polio vaccine (DTaP-IPV) at 4 years of age. Cell-mediated immune responses to pertussis vaccine antigens were measured at 2 years of age. Adverse events following DTaP-IPV were recorded. Results: Of 74 subjects, 52 (70%) were available for follow up. Overall, 11 (21%) had detectable PT IgG at 2 years, decreasing to 10% before 4-year-old booster compared with 100% at 8 months of age. After the 4-year booster, pertussis antigen IgG levels were similar, but there was a trend to lower PT IgG levels in birth aP infants (geometric mean concentrations: 28.7 EI.U/mL) compared with controls (geometric mean concentrations: 53.6 EI.U/mL). The cytokine responses to pertussis antigen stimulation were higher in aP recipients at 2 years of age. There was no difference in injection site reactions among groups following the DTaP-IPV booster at 4 years of age. Conclusions: In the longest reported follow-up of infants who received aP vaccine at birth, we found a trend to lower PT IgG antibodies post booster compared with receipt of first dose of aP-containing vaccine at 8 weeks of age. Short- and long-term antibody responses with and without prior maternal pertussis vaccination are crucial for further evaluation of this strategy for preventing severe early pertussis.