Altered Fear Behavior in Aeroallergen House Dust Mite Exposed C57Bl/6 Mice: A Model of Th2-skewed Airway Inflammation.

There is a growing interest for studying the impact of chronic inflammation, particularly lung inflammation, on the brain and behavior. This includes asthma, a chronic inflammatory condition, that has been associated with psychiatric conditions such as posttraumatic stress disorder (PTSD). Although asthma is driven by elevated production of Th2 cytokines (IL-4, IL-5 and IL-13), which drive asthma symptomology, recent work demonstrates that concomitant Th1 or Th17 cytokine production can worsen asthma severity. We previously demonstrated a detrimental link between PTSD-relevant fear behavior and allergen-induced lung inflammation associated with a mixed Th2/Th17-inflammatory profile in mice. However, the behavioral effects of Th2-skewed airway inflammation, typical to mild/moderate asthma, are unknown. Therefore, we investigated fear conditioning/extinction in allergen house dust mite (HDM)-exposed C57Bl/6 mice, a model of Th2-skewed allergic asthma. Behaviors relevant to panic, anxiety, and depression were also assessed. Furthermore, we investigated the accumulation of Th2/Th17-cytokine-expressing cells in lung and brain, and the neuronal activation marker, ΔFosB, in fear regulatory brain areas. HDM-exposed mice elicited lower freezing during fear extinction with no effects on acquisition and conditioned fear. No HDM effect on panic, anxiety or depression-relevant behaviors was observed. While HDM evoked a Th2-skewed immune response in lung tissue, no significant alterations in brain Th cell subsets were observed. Significantly reduced ΔFosB+ cells in the basolateral amygdala of HDM mice were observed post extinction. Our data indicate that allergen-driven Th2-skewed responses may induce fear extinction promoting effects, highlighting beneficial interactions of Th2-associated immune mediators with fear regulatory circuits.

Modulation of fear behavior and neuroimmune alterations in house dust mite exposed A/J mice, a model of severe asthma.

Fear-associated conditions such as posttraumatic stress disorder (PTSD) and panic disorder (PD) are highly prevalent. There is considerable interest in understanding contributory risk and vulnerability factors. Accumulating evidence suggests that chronically elevated inflammatory load may be a potential risk factor for these disorders. In this regard, an association of asthma, a chronic inflammatory condition, with PTSD and PD has been reported. Symptoms of PD and PTSD are more prevalent in severe asthmatics, compared to those with mild or moderate asthma suggesting that factors that influence the severity of asthma, may also influence susceptibility to the development of fear-related disorders. There has been relatively little progress in identifying contributory factors and underlying mechanisms, particularly, the translation of severe asthma-associated lung inflammation to central neuroimmune alterations and behavioral manifestations remains unclear. The current study investigated the expression of behaviors relevant to PD and PTSD (CO2 inhalation and fear conditioning/extinction) in A/J mice using a model of severe allergic asthma associated with a mixed T helper 2 (Th2) and Th17 immune response. We also investigated the accumulation of Th2- and Th17-cytokine expressing cells in lung and brain tissue, microglial alterations, as well as neuronal activation marker, delta FosB (ΔFosB)) in fear and panic regulatory brain areas. HDM-exposed mice elicited higher freezing during fear extinction. CO2-associated spontaneous and conditioned freezing, as well as anxiety or depression-relevant exploratory and coping behaviors were not altered by HDM treatment. A significant increase in brain Th17-associated inflammatory mediators was observed prior to behavioral testing, accompanied by microglial alterations in specialized blood brain barrier-compromised circumventricular area, subfornical organ. Post extinction measurements revealed increased ΔFosB staining within the medial prefrontal cortex and basolateral amygdala in HDM-treated mice. Collectively, our data show modulation of brain immune mechanisms and fear circuits by peripheral airway inflammation, and is relevant to understanding the risk and comorbidity of asthma with fear-associated disorders such as PTSD.

IL-21 receptor signalling partially mediates Th2-mediated allergic airway responses.

BACKGROUND: Interleukin-21 (IL-21) has been implicated in the development of Th2-mediated immune responses; however, the exact role it plays in allergic diseases is not well understood. OBJECTIVE: To elucidate the contribution of IL-21 receptor signalling to Th2-dependent immune responses in the lung. METHODS: We compared allergic airway responses in wild-type BALB/c and Il21r-deficient mice exposed to local airway challenge with house dust mite (HDM). RESULTS: We demonstrate that IL-21R-deficiency reduces HDM-driven airway hyperresponsiveness (AHR) with only partial effects on airway inflammation. Concomitant with the reduction in AHR in Il21r-deficient mice, significant suppression was observed in protein levels of the Th2 cytokines IL-4, and IL-13. In contrast, IL-21R-deficiency was associated with an increase in PBS- and allergen-driven IgE levels, while IgG1 and IgG2a levels were decreased. Moreover, our results suggest that IL-21 may contribute to AHR through its ability to both directly induce Th2 cell survival and to impair regulatory T-cell suppression of Th2 cytokine production. Importantly, we show that IL-21-positive cells are increased in the bronchial mucosa of asthmatics compared with non-asthmatics. CONCLUSION: These results suggest that IL-21 plays an important role in the allergic diathesis by enhancing Th2 cytokine production through multiple mechanisms including the suppression of Treg inhibitory effects on Th2 cell cytokine production.

PD-L2 modulates asthma severity by directly decreasing dendritic cell IL-12 production.

Studies examining the role of programmed death 1 (PD-1) ligand 2 (PD-L2)/PD-1 in asthma have yielded conflicting results. To clarify its role, we examined the PD-L2 expression in biopsies from human asthmatics and the lungs of aeroallergen-treated mice. PD-L2 expression in bronchial biopsies correlated with the severity of asthma. In mice, allergen exposure increased PD-L2 expression on pulmonary myeloid dendritic cells (DCs), and PD-L2 blockade diminished allergen-induced airway hyperresponsiveness (AHR). By contrast, PD-1 blockade had no impact, suggesting that PD-L2 promotes AHR in a PD-1-independent manner. Decreased AHR was associated with enhanced serum interleukin (IL)-12 p40, and in vitro stimulation of DCs with allergen and PD-L2-Fc reduced IL-12 p70 production, suggesting that PD-L2 inhibits allergen-driven IL-12 production. In our model, IL-12 did not diminish T helper type 2 responses but rather directly antagonized IL-13-inducible gene expression, highlighting a novel role for IL-12 in regulation of IL-13 signaling. Thus, allergen-driven enhancement of PD-L2 signaling through a PD-1-independent mechanism limits IL-12 secretion, exacerbating AHR.

Dendritic cell-derived tumor necrosis factor α modifies airway epithelial cell responses.

Mucosal dendritic cells (DC) are intimately associated with the airway epithelium and thus are ideally situated to be first responders to pathogens. We hypothesize that DC drive innate immune responses through early release of tumor necrosis factor (TNF) α, which drives airway epithelial cell responses. In a mouse model, TNFα release was significantly increased following a single exposure to German cockroach (GC) frass, an event independent of neutrophil recruitment into the airways. While lung epithelial cells and alveolar macrophages failed to release TNFα following GC frass exposure, bone marrow-derived DC (BMDC) produced substantial amounts of TNFα suggesting their importance as early responding cells. This was confirmed by flow cytometry of pulmonary myeloid DC. Addition of GC frass-pulsed BMDC or conditioned media from GC frass-pulsed BMDC to primary mouse tracheal epithelial cells (MTEC) or MLE-15 cells induced chemokine (C-C) motif ligand (CCL) 20 and granulocyte macrophage (GM) colony-stimulating factor (CSF), both of which are important for DC recruitment, survival and differentiation. Importantly, DC do not produce CCL20 or GM-CSF following allergen exposure. Blocking TNFα receptor 1 (TNFR1) completely abolished chemokine production, suggesting that BMDC-derived TNFα induced airway epithelial cell activation and enhancement of the innate immune response. Lastly, blocking TNFR1 in vivo resulted in significantly decreased CCL20 and GM-CSF production in the lungs of mice. Together, our data strongly suggest that DC-derived TNFα plays a crucial role in the initiation of innate immune responses through the modification of airway epithelial cell responses.

Cytokine-based immunotherapy of allergic disease.

Human immediate hypersensitivity diseases are strongly associated with an excessive type 2 response to normally innocuous environmental antigens, and are a growing health care concern in developed nations. Commonly prescribed treatments provide effective symptomatic relief, but are unable to consistently ameliorate the underlying cause of allergic disease: the excessive generation of allergen-specific Th2 cells. IL-12 and IL-18 are potent inducers of type 1 immunity, and, as such, have been proposed as candidates for treatment of allergic diseases. This review critically assesses the potential of recombinant IL-12 and IL-18 immunotherapy to redirect both de novo and established allergic responses in animal models of human allergic disease to clinically protective immune responses.

Comparison of chemiluminescent assays and colorimetric ELISAs for quantification of murine IL-12, human IL-4 and murine IL-4: chemiluminescent substrates provide markedly enhanced sensitivity.

Many available ELISAs lack the sensitivity required to reliably quantify levels of cytokines released in response to antigenic stimulation. In an effort to increase sensitivity of these assays, we compare the sensitivity of standard colorimetric ELISAs and corresponding chemiluminescent assays for three cytokines which are usually produced in very low quantities: mouse IL-12 p70, human IL-4 and mouse IL-4. Use of a chemiluminescent substrate enhanced the sensitivity of these assays 12-29-fold as compared to current colorimetric ELISAs. Accompanying this increase in sensitivity was an increase in dynamic range, a decrease in the time required to obtain maximum sensitivity and a decrease in the concentration of reagents required. These findings are of particular interest to those wanting to quantitate levels of any cytokine which is typically produced in low levels.

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-gamma 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.