Research reportNLRP3 gene knockout blocks NF-κB and MAPK signaling pathway in CUMS-induced depression mouse model
Introduction
Major depressive disorder (MDD) characterizes by depressed mood most of the day or markedly diminished interest or pleasure in all activities most of the day [1]. As a pervasive mental disorder and major contributor to the global burden of diseases, there are about 350 million people suffering from MDD across the world [2], [3]. The prevalence of MDD for lifetime was 16.2% in US (32.6-35.1 million US adults), and the overall estimation of lifetime prevalence of MDD was 3.3% in mainland of China [4], [5]. As it has huge social and economic importance, researchers around the world are trying to figure out the fundamental pathophysiological mechanisms of MDD. After decades of extensive research, researchers postulate several hypotheses potentially underlying the MDD progression, such as the neurotransmitter hypothesis, neuronal circuit hypothesis and proinflammatory cytokine hypothesis.
The proinflammatory cytokine hypothesis suggests that inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 and interleukin-1β (IL-1β), contribute to neuroinflammation and suppress neurogenesis in the central nervous system [6], [7], [8], [9]. Reichenberg A et al. reported that in human, endotoxin administration resulted in transient significant increase in the levels of anxiety and depressed mood [10], while Goshen et al. found that in mice, elevated levels of brain IL-1 were causally related to various aspects of depression-like behavior, including the behavioral symptomatology, adrenocortical activation and reduced neurogenesis [11]. It is notable that maturation and secretion of IL-1β is dependent upon the activation of NLRP3 inflammasome, which is a multi-protein complex of the innate immune system, and serves as an upstream regulator of the IL-1β signaling pathway [12], [13], [14]. Iwata et al. postulated that the NLRP3 inflammasome might be a novel target for the development of treatments for MDD [15]. Our previous studies suggested that the NLRP3 inflammasome mediated lipopolysaccharide (LPS) and chronic unpredictable mild stress (CUMS) induced depression-like behaviors in mice [16], [17]. Alcocer-Gomez et al. indicated that stress-induced depression-like behaviors required a functional NLRP3 inflammasome [18].
There are three major models for NLRP3 inflammasome activation: (1) NLRP3 inflammasome agonist adenosine triphosphate (ATP) triggers activation of the purinergic type 2X7 receptor (P2X7R) and leads other extracellular agonists to entering the cytosol; (2) Danger-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs) lead to NLRP3 inflammasome formation via the reactive oxygen species (ROS) dependent pathway; (3) The engulfed crystalline or particulate NLRP3 inflammasome agonists can be sensed in the cytoplasm after lysosomal rupture [12]. Iwata et al. reported that the ATP/P2X7R-NLRP3 inflammasome cascade in the brain could sense the psychological stress, which led to anhedonic and anxious behaviors [19]. Our previous study found that blocking generation of ROS in the hippocampus and prefrontal cortex (PFC) with antioxidative hydrogen-rich water could inhibit inflammasome activation and prevent the development of depression-like behaviors induced by CUMS [20].
As an important contributor to the pathogenesis of MDD, ROS might exert effects on inflammatory pathways via activating the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinases (MAPK) family stress kinases [21]. Bioinformatics analysis found that the MAPK pathway was one of the functionally enriched cell-signaling pathways engaged in MDD [22]. Since NF-κB could interact with a bunch of genes involved in inflammation, researchers observed activation of NF-κB in many inflammatory diseases, such as arthritis, asthma and atherosclerosis [23]. Song et al. found that NF-κB was associated with schizophrenia through regulating the expression of cytokines in patients’ serum [24]. Several studies indicated that the NF-κB signaling cascade might have particular relevance to MDD [21], [25], [26]. In an integrative rat-human study identifying genes and gene pathways associated with MDD, researchers found that eighty percent of those uncovered genes that might have a role in the pathogenesis of MDD were functionally associated with stress response signaling cascade, involving NF-κB and MAPK pathway [27].
However, it is not clear if and how the NLRP3 inflammasome influences the NF-κB and MAPK pathway to contribute to neuroinflammation in MDD. We hypothesize that the NLRP3 inflammasome pathway may exert its effects on MDD via regulating the NF-κB and MAPK stress response signaling cascade. In this study, both wild-type and NLRP3 gene knockout mice are subjected to CUMS treatment. After 4-week stress exposure, we perform depression-like behavior tests and measure IL-1β levels, NLRP3 expression, NF-κB activation and MAPK pathway protein expression to explore if and how NLRP3 gene knockout influence the NF-κB and MAPK inflammatory pathway in MDD animal model. As we expected, the results suggested that the NLRP3 inflammasome contributed to neuroinflammation through regulating NF-κB and MAPK inflammatory signaling pathways in MDD.
Section snippets
Experimental animals
Male wild-type 8-week-old and age-matched male NLRP3 gene knockout C57BL/6 mice (NLRP3 KO) were all introduced from New Drug Safety Evaluation Center, Second Military Medical University (Shanghai, China) [28], [29]. Mice were housed in a standardized animal room (lights on 7a.m.–7p.m.; room temperature 22 ± 2 °C), with food and water provided ad libitum. Before stress procedures, mice were acclimated to the environment and 1% sucrose solution (weight/volume) for 14 days and then randomly assigned
Polyacrylamide gel electrophoresis-based genotyping analysis
In order to genotype NLRP3 gene knockout mice, a polyacrylamide gel electrophoresis-based method was applied. F2 generation homozygous NLRP3 knockout mice were adopted during the mating process of F1 generation heterozygote mice and genotyped by T7E1 digestion method. Based on NLRP3 PCR primers and mutant positions, different DNA pattern occurred after T7E1 digestion: wild-type band of 366 bp; mutant band of approximately 226 + 140 bp. NLRP3 F2 3–9 was homozygous and occurred only one band after
Discussion
In our previous studies, we found that the NLRP3 inflammasome signaling pathway inhibitors Ac-YVAD-CMK and VX-765 could impede LPS-induced acute depression-like behaviors and CUMS-induced chronic depression-like behaviors respectively [16], [17]. To further investigate the functional role of NLRP3 inflammasome in CUMS-induced depression-like behaviors and the relevant inflammatory signaling pathways, we compared the behavioral and molecular parameters of wild-type and NLRP3 gene knockout mice.
Conclusions
In summary, these results further demonstrated that the NLRP3 inflammasome was involved in the CUMS-induced depression-like behaviors via regulating IL-1β protein expression in serum and hippocampus. The NLRP3 inflammasome could influence the NF-κB signaling pathway and MAPK family stress kinases signaling pathway. These two pathways might mediate the downstream inflammatory effects of NLRP3 inflammasome. Our study suggested that interventions targeting the inflammasome-IL-1β signaling
Author’s contributions
W-JS and YZ (Yi Zhang) contributed equally to this work. C-LJ and G-CL designed the research; S-JF and YZ bred the NLRP3 gene knockout mice. W-JS and YZ (Yi Zhang) conducted the establishment of depression mouse model; YC, HG and Y-JL assisted behavioral measurement and sample collection; YZ (Yi Zhang) and YC detected IL-1β levels in serum and hippocampi; W-JS detected MAPK pathway protein expression and NF-κB translocation.; WP detected the IκB-α and NLRP3 protein expression. YZ (Yi Zhang) and
Conflict of interest
The authors declare no conflict of interest.
Acknowledgements
This study funded by the National Natural Science Foundation of China (81571169, 31371200 to C-LJ), the National Instrumentation Program of China (2013YQ190467 to C-LJ), and the Military Medical Research Project (BWS14J021 to C-LJ).
References (47)
- et al.
Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions, 1990–2010: a systematic analysis for the Global Burden of Disease Study 2010
Lancet
(2012) Evidence for an immune response in major depression: a review and hypothesis
Prog. Neuro-Psychopharmacol. Biol. Psychiatry
(1995)- et al.
Immune suppression and immune activation in depression
Brain Behav. Immun.
(2011) - et al.
A meta-analysis of cytokines in major depression
Biol. Psychiatry
(2010) - et al.
The inflammasomes
Cell
(2010) - et al.
The NLRP3 inflammasome: a sensor of immune danger signals
Semin. Immunol.
(2009) - et al.
Nlrp3: an immune sensor of cellular stress and infection
Int. J. Biochem. Cell Biol.
(2010) - et al.
The inflammasome: pathways linking psychological stress, depression, and systemic illnesses
Brain Behav. Immun.
(2013) - et al.
Psychological stress activates the inflammasome via release of adenosine triphosphate and stimulation of the purinergic type 2 × 7 receptor
Biol. Psychiatry
(2016) - et al.
The interaction of nuclear factor-kappa B and cytokines is associated with schizophrenia
Biol. Psychiatry
(2009)
Activation of the NLRP3 inflammasome in lipopolysaccharide-induced mouse fatigue and its relevance to chronic fatigue syndrome
J. Neuroinflamm.
Interleukin-1 (IL-1): a central regulator of stress responses
Front. Neuroendocrinol.
Anti-inflammatory compounds parthenolide and Bay 11-7082 are direct inhibitors of the inflammasome
J. Biol. Chem.
NF-kappa B: ten years after
Cell
Symmetrical treatment of diagnostic and statistical manual of mental disorders, fifth edition, for major depressive disorders
Source Code Biol. Med.
The Global Burden of Disease 2004 Update
Epidemiology of major depressive disorder in mainland china: a systematic review
PLoS One
The epidemiology of major depressive disorder: results from the National Comorbidity Survey Replication (NCS-R)
JAMA
Inflammatory aspects of depression
Inflamm. Allergy Drug Targets
Cytokine-associated emotional and cognitive disturbances in humans
Arch. Gen. Psychiatry
Brain interleukin-1 mediates chronic stress-induced depression in mice via adrenocortical activation and hippocampal neurogenesis suppression
Mol. Psychiatry
NLRP3 inflammasome mediates chronic mild stress-induced depression in mice via neuroinflammation
Int. J. Neuropsychopharmacol.
Involvement of inflammasome activation in lipopolysaccharide-induced mice depressive-like behaviors
CNS Neurosci. Ther.
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These authors contributed equally to this work.