Correction to: HSP60 critically regulates endogenous IL-1ß production in activated microglia by stimulating NLRP3 inflammasome pathway.

Upon publication of the original article [1], it was noticed that there is an error in Fig. 10, the dialog box in panel (b) was missing. The correct Fig. 10 is shown below.

HSP60 critically regulates endogenous IL-1ß production in activated microglia by stimulating NLRP3 inflammasome pathway.

BACKGROUND: Interleukin-1ß (IL-1ß) is one of the most important cytokine secreted by activated microglia as it orchestrates the vicious cycle of inflammation by inducing the expression of various other pro-inflammatory cytokines along with its own production. Microglia-mediated IL-1ß production is a tightly regulated mechanism which involves the activation of nucleotide-binding oligomerization domain leucine-rich repeat and pyrin domain-containing 3 (NLRP3) inflammasome pathway. Our previous study suggests the critical role of heat shock protein 60 (HSP60) in IL-1ß-induced inflammation in microglia through TLR4-p38 MAPK axis. However, whether HSP60 regulates endogenous IL-1ß production is not known. Therefore, to probe the underlying mechanism, we elucidate the role of HSP60 in endogenous IL-1ß production. METHODS: We used in vitro (N9 murine microglial cells) and in vivo (BALB/c mouse) models for our study. HSP60 overexpression and knockdown experiment was done to elucidate the role of HSP60 in endogenous IL-1ß production by microglia. Western blotting and quantitative real-time PCR was performed using N9 cells and BALB/c mice brain, to analyze various proteins and transcript levels. Reactive oxygen species levels and mitochondrial membrane depolarization in N9 cells were analyzed by flow cytometry. We also performed caspase-1 activity assay and enzyme-linked immunosorbent assay to assess caspase-1 activity and IL-1ß production, respectively. RESULTS: HSP60 induces the phosphorylation and nuclear localization of NF-κB both in vitro and in vivo. It also induces perturbation in mitochondrial membrane potential and enhances reactive oxygen species (ROS) generation in microglia. HSP60 further activates NLRP3 inflammasome by elevating NLRP3 expression both at RNA and protein levels. Furthermore, HSP60 enhances caspase-1 activity and increases IL-1ß secretion by microglia. Knockdown of HSP60 reduces the IL-1ß-induced production of IL-1ß both in vitro and in vivo. Also, we have shown for the first time that knockdown of HSP60 leads to decreased IL-1ß production during Japanese encephalitis virus (JEV) infection, which eventually leads to decreased inflammation and increased survival of JEV-infected mice. CONCLUSION: HSP60 mediates microglial IL-1ß production by regulating NLRP3 inflammasome pathway and reduction of HSP60 leads to reduction of inflammation in JEV infection.

HSP60 plays a regulatory role in IL-1ß-induced microglial inflammation via TLR4-p38 MAPK axis.

BACKGROUND: IL-1ß, also known as "the master regulator of inflammation", is a potent pro-inflammatory cytokine secreted by activated microglia in response to pathogenic invasions or neurodegeneration. It initiates a vicious cycle of inflammation and orchestrates various molecular mechanisms involved in neuroinflammation. The role of IL-1ß has been extensively studied in neurodegenerative disorders; however, molecular mechanisms underlying inflammation induced by IL-1ß are still poorly understood. The objective of our study is the comprehensive identification of molecular circuitry involved in IL-1ß-induced inflammation in microglia through protein profiling. METHODS: To achieve our aim, we performed the proteomic analysis of N9 microglial cells with and without IL-1ß treatment at different time points. Expression of HSP60 in response to IL-1ß administration was checked by quantitative real-time PCR, immunoblotting, and immunofluorescence. Interaction of HSP60 with TLR4 was determined by co-immunoprecipitation. Inhibition of TLR4 was done using TLR4 inhibitor to reveal its effect on IL-1ß-induced inflammation. Further, effect of HSP60 knockdown and overexpression were assessed on the inflammation in microglia. Specific MAPK inhibitors were used to reveal the downstream MAPK exclusively involved in HSP60-induced inflammation in microglia. RESULTS: Total 21 proteins were found to be differentially expressed in response to IL-1ß treatment in N9 microglial cells. In silico analysis of these proteins revealed unfolded protein response as one of the most significant molecular functions, and HSP60 turned out to be a key hub molecule. IL-1ß induced the expression as well as secretion of HSP60 in extracellular milieu during inflammation of N9 cells. Secreted HSP60 binds to TLR4 and inhibition of TLR4 suppressed IL-1ß-induced inflammation to a significant extent. Our knockdown and overexpression studies demonstrated that HSP60 increases the phosphorylation of ERK, JNK, and p38 MAPKs in N9 cells during inflammation. Specific inhibition of p38 by inhibitors suppressed HSP60-induced inflammation, thus pointed towards the major role of p38 MAPK rather than ERK1/2 and JNK in HSP60-induced inflammation. Furthermore, silencing of upstream modulator of p38, i.e., MEK3/6 also reduced HSP60-induced inflammation. CONCLUSIONS: IL-1ß induces expression of HSP60 in N9 microglial cells that further augments inflammation via TLR4-p38 MAPK axis.

MicroRNA 155 regulates Japanese encephalitis virus-induced inflammatory response by targeting Src homology 2-containing inositol phosphatase 1.

UNLABELLED: MicroRNAs (miRNAs) are single-stranded small RNA molecules that regulate various cellular processes. miRNA 155 (miR-155) regulates various aspects of innate and adaptive immune responses and plays a key role in various viral infections and the resulting neuroinflammation. The present study evaluated the involvement of miR-155 in modulating Japanese encephalitis virus (JEV)-induced neuroinflammation. We observed that miR-155 expression was upregulated during JEV infection of mouse primary microglia, the BV-2 microglia cell line, and in both mouse and human brains. In vitro and in vivo knockdown of miR-155 minimized JEV-induced inflammatory responses. In the present study, we confirmed targeting of the Src homology 2-containing inositol phosphatase 1 (SHIP1) 3' untranslated region (UTR) by miR-155 in the context of JEV infection. Inhibition of SHIP1 by miR-155 resulted in higher beta interferon (IFN-ß) and proinflammatory cytokine production through activation of TANK-binding kinase 1 (TBK-1). Based on these observations, we conclude that miR-155 modulates the neuroinflammatory response during JEV infection via negative regulation of SHIP1 expression. Thus, modulation of miR-155 could be a novel strategy to regulate JEV-induced neuroinflammation. IMPORTANCE: Japanese encephalitis virus (JEV), a member of the family Flaviviridae that causes Japanese encephalitis (JE), is the most common mosquito-borne encephalitis virus in the Asia-Pacific region. The disease is feared, as currently there are no specific antiviral drugs available. JEV targets the central nervous system, leading to high mortality and neurological and psychiatric sequelae in some of those who survive. The level of inflammation correlates well with the clinical outcome in patients. Recently, microRNA (miRNA), a single-stranded noncoding RNA, has been implicated in various brain disorders. The present study investigates the role of miRNA in JEV-induced neuroinflammation. Our results show that miRNA 155 (miR-155) targets the Src homology 2-containing inositol phosphatase 1 (SHIP1) protein and promotes inflammation by regulating the NF-κB pathway, increasing the expression of various proinflammatory cytokines and the antiviral response. Thus, miR-155 is a potential therapeutic target to develop antivirals in JE and other brain disorders where inflammation plays a significant role in disease progression.