Background
Asthma is the most common chronic airway disease that has implied a much greater prevalence of mental disorders such as bipolar disorder (BD) [
1,
2]. The association of asthma and psychiatric comorbidity indicated symptom severity [
3,
4], poorer asthma control [
5], and higher comorbidities and increased use of health services [
6], leading to heavy socioeconomic burden [
6,
7]. Thus, this clinical phenomenology has caused greater attention and concern from public health communities worldwide. However, to the best of our knowledge, the mechanism about the association between asthma and BD is not unequivocal.
Airway inflammation is the most important pathological feature of asthma [
8], featuring the increased migration and activation of Th2 lymphocytes, mast cells, eosinophils, and macrophages [
9,
10]. Inflammatory cytokines like interleukin-4 (IL-4), IL-5, and IL-13 are also increased and altered along with asthma exacerbation [
11]. Notably, accumulating evidences have proved that dysfunction of inflammation occur in mental disorder patients [
12]. IL-4, IL-6, and IL-12 may change in bipolar disorder (BD) patients under different mood episodes (mania or depression episode) [
13,
14]. More and more related studies indicate that the immune system responses chronically activated by macrophages and T lymphocytes may result in mood dysregulation as the peripheral inflammation transmits information to the brain [
15]. Consistent with this notion, microglia, the resident immune cells in the brain, might function as an important interface to transmit such information [
16]. Moreover, peripheral immune system and inflammatory processes have demonstrated alteration in many patients with bipolar disorder [
17]. Cytokines, connecting peripheral immune to central nervous systems [
15], have shown altered levels in patients with bipolar disorder as compared with individuals without disorder [
17]. However, few reports have examined the direct relevance between airway inflammation and its psychiatric comorbidity BD in asthma.
Our previous work found that ITGB4, a structural adhesion molecule, is downregulated in airway epithelial cells of asthma patients with four variation sites in 5′ flanking region [
18,
19]. ITGB4, a heterodimeric transmembrane receptor, is located at the basal surface of airway epithelial cells in hemidesmosomal structures that function as structural link between epidermal cells and the underlying basement membrane [
20,
21]. ITGB4 also regulates pathological airway conditions of inflammation responses through integrin-associated signaling and recruitment of adaptor molecules [
22,
23]. In addition, ITGB4 leads to activation of the Rho GTPases [
24] as well as MAPK [
25], PI3-K [
26], and NF-κB signaling [
27] pathways which were highly relevant to the propagation of inflammation and injury. Meanwhile, the increased permeability of the bronchial epithelium to HDM has been associated with enhanced NF-κB activity and increased pro-inflammatory cytokine expression, which implies that the disruptions of airway epithelial barrier may have immunomodulatory consequences [
28,
29].
Therefore, in the present manuscript, we utilized ITGB4 conditional knockout mice to investigate the direct induction of airway inflammation with BD and unravel the mechanisms that peripheral inflammation information is transmitted to the brain to affect the neuronal network and trigger BD-like behaviors.
Discussion
The present study verified that airway inflammatory is the important incentive for the BD-like behavior during asthma pathogenesis. ITGB4 seems to be a critical participant in the induction of airway inflammation and BD-related behavior. Bronchial epithelial ITGB4 knocked out from the embryonic stage could induce systematically chronic inflammation, microglial activation to secrete neuro-inflammatory cytokines probably through the circumventricular organs and the choroid plexus, and further pathophysiologic changes in the brain to result in BD-like behavior. These findings may help to provide a new animal model for studying the comorbidity of asthma and BD as well as a new avenue for treatment.
Accumulating clinical studies showed that there was significantly high prevalence of BD in asthmatic patients. However, few animal models were used to study the linkages and underlying mechanism between asthma and BD. It is interesting that bronchial epithelial ITGB4 conditional knockout mice presented BD-like behavior obviously. In behavioral test, ITGB4
−/− mice showed an increased locomotor activity and decreased habituation in the open-field test, which are typical symptoms of mania and ADHD [
39,
41]. Several clinical studies observed that bipolar patients exhibit a greater response to D-AMPH [
42], showing increased activity in manic episodes of BD [
43], while ADHD patients exert calming response to D-AMPH [
44]. As expected, ITGB4
−/− mice treated with D-AMPH in the present study showed increased locomotor activity, indicating that the enhanced sensitivity of ITGB4
−/− mice to
d-amphetamine is related with mania rather than ADHD.
Moreover, the behavioral analysis revealed increased exploration as measured by center time duration in the 5-min open-field test in ITGB4
−/− mice. Besides increased exploration, the increased time spent in the center in ITGB4
−/− mice, as well as increased entry into and time in the open arms in the elevated plus maze [
45] could be related to the mice’s reduced anxiety/increased risking behavior [
46,
47], a mania-linked characteristic in manic patients [
48,
49]. These findings provide further consistency to increased object interactions in patients with BD [
47].
To define the depressive-like behavior of ITGB4
−/− mice, we utilized the forced swim test, one of the most commonly used model of behavioral despair. We found an increased immobility time in ITGB4
−/− mice, indicating depressive-like behavior in these mice. Anhedonia, a decreased ability to experience pleasure, is also a symptom of depressive-like behavior [
50,
51], and reduced sucrose consumption is usually used as an indicator of depression-related anhedonia [
51]. Although no significant difference was found in sucrose preference between ITGB4
−/− mice and WT littermates, the sucrose consumption decreased after restraint stress in ITGB4
−/− mice. Stress plays an acknowledged role in precipitating psychotic episodes in bipolar disorder [
52]. Many stressors including psychological, hormonal, and pharmacological that induce modest, transient perturbations in healthy individuals are able to induce mood episodes in individuals with bipolar diathesis [
53,
54]. To further affirm this behavioral phenotype, learned helplessness test, an important model to measure helplessness or despair involving sub-chronic stress, was conducted on ITGB4
−/− mice and WT littermates. We found that ITGB4
−/− mice in the FS group show more escape failures than control animals, suggesting that ITGB4
−/− mice are more impaired in learned helplessness. These findings indicate that the ITGB4
−/− mice are impressionable to the depression effects of stress.
This study detects hyperlocomotion, psychostimulants sensitivity, anxiolytic-like behavior, and stress-induced depression in ITGB4
−/− mice, which were analogous to some features of human BD characterized by an episodic recurrent pathological disturbance in mood ranging from severe depression to extreme mania [
55]. In previous reports, evidences reveal that both asthma and BD share common immune dysfunction [
1,
56]. And ITGB4 deficiency may play an important role in airway inflammation of asthma patients [
19]. Exhilaratedly, our findings revealed that ITGB4
−/− mice predominantly exhibited Th2-type inflammation, which was the pivotal characteristic of asthma [
57]. IL-4 and IL-13, the major cytokine of Th2-type inflammation, were very highly expressed in bronchoalveolar lavage fluid and blood of ITGB4
−/− mice. Meanwhile, IL-4 was also reported elevated immune activation in both manic and depressive state [
14,
58]. Combined with previous experimental results that ITGB4
−/− mice presented BD-like behavior in our study, these findings verified that Th2 inflammation played a critical role in the association between asthma and BD in ITGB4
−/− mice.
As we all know, the effect of IL-4 signaling is mediated through the IL-4 receptor alpha chain (IL-4Rα), which dimerizes either with the common gamma chain (CD132) or with the IL-13 receptor alpha 1 (IL-13Rα1) chain [
59]. A number of cell types including macrophages, neurons, astrocytes, and microglia express the IL-4Rα and can respond to IL-4 signaling [
59,
60]. In ITGB4
−/− mice, IL-4Rα was found highly expressed in choroid plexus (CP), an important area of circumventricular organs (CVOs) with leaky blood–brain barriers (BBBs). Circumventricular organ macrophages responding to IL-4 could release pro-inflammatory cytokine, which then leakage into the brain to promote the production of a second wave of cytokines by microglial cells [
61]. Microglia, resident macrophage-like immune cells in the CNS, play a critical role in both physiological and pathological conditions, including restoring the homeostasis of the CNS and driving the neuro-inflammatory response of neurodegenerative disorders, respectively [
62]. We really found that ITGB4
−/− mice showed a significant increase of microglial and pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α.
Furthermore, some researches have implicated immune factors in brain development and plasticity [
63]. Microglia promote pro-inflammatory responses with excess tumor necrosis factor (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS) production [
64], contributing to neural network dysfunction. Thus, it is possible that inflammation and immune activation could affect brain regions involved in the progress and variation in symptom levels in bipolar disorder. IL-1β is widely distributed in the brain, particularly in the hippocampus and hypothalamus [
65]. Consistent with previous results, IL-1β was highly expressed in the hippocampus in ITGB4
−/− mice. Subsequent animal studies have indicated that high levels of IL-1β can act on hippocampal neurons to inhibit synaptic strengthening and LTP [
66,
67], which was conformed by the decrease of SYP in ITGB4
−/− mice. A postmortem study on the prefrontal cortex revealed that the IL-1β protein and mRNA levels were significantly higher in the patients with BD [
68]. This result is in line with our findings that IL-1β expression increased obviously in the prefrontal cortex of ITGB4
−/− mice. In the last decade, the importance of cytokines in neuronal survival [
69] was recognized in the pathophysiology of BD. ITGB4
−/− mice did show neuron damage and increased neuron apoptosis. In addition, many studies have also reported increased TNF-α and IL-6 levels in acute phases of mania and depression compared to the controls [
14,
58,
70]. Pro-inflammatory cytokine TNF-α and IL-6 in neurotransmitters, neuroplasticity, and neuronal survival [
71‐
73] was linked to the pathophysiology of BD. Interestingly, our experiments showed that the expression levels of TNF-α and IL-6 were higher in hippocampus and prefrontal cortex of the ITGB4
−/− mice than the control mice. These results support the concept that BD have been associated with changes of the immune response.