BackgroundIL-1β, 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-1β has been extensively studied in neurodegenerative disorders; however, molecular mechanisms underlying inflammation induced by IL-1β are still poorly understood. The objective of our study is the comprehensive identification of molecular circuitry involved in IL-1β-induced inflammation in microglia through protein profiling.MethodsTo achieve our aim, we performed the proteomic analysis of N9 microglial cells with and without IL-1β treatment at different time points. Expression of HSP60 in response to IL-1β 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-1β-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.ResultsTotal 21 proteins were found to be differentially expressed in response to IL-1β 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-1β 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-1β-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.ConclusionsIL-1β induces expression of HSP60 in N9 microglial cells that further augments inflammation via TLR4-p38 MAPK axis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0486-x) contains supplementary material, which is available to authorized users.

Japanese encephalitis virus (JEV), which is a causative agent of sporadic encephalitis, harbours itself inside the neural stem/progenitor cells. It is a well-known fact that JEV infects neural stem/progenitor cells and decreases their proliferation capacity. With mass spectrometry-based quantitative proteomic study, it is possible to reveal the impact of virus on the stem cells at protein level. Our aim was to perceive the stem cell proteomic response upon viral challenge. We performed a two-dimensional gel electrophoresis-based proteomic study of the human neural stem cells (hNS1 cell line) post JEV infection and found that 13 proteins were differentially expressed. The altered proteome profile of hNS1 cell line revealed sustained endoplasmic reticulum stress, which deteriorated normal cellular activities leading to cell apoptosis. The proteomic changes found in hNS1 cell line were validated in vivo in the subventricular zone of JE infected BALB/c mice. Congruent alterations were also witnessed in multipotent neural precursor cells isolated from human foetus and in autopsy samples of human brain clinically diagnosed as cases of JE patients. Endoplasmic reticulum resident chaperone GRP78, mitochondrial protein Prohibitin and heterogeneous nuclear ribonucleoprotein hnRNPC (C1/C2) have been shown to interact with viral RNA. Hence it is proposed that these are the principle candidates governing endoplasmic reticulum stress-induced apoptosis in JEV infection.

Clostridium perfringens is a medically important clostridial pathogen and an etiological agent causing several diseases in humans and animals. C. perfringens and its toxins have been listed as potential biological and toxin warfare (BTW) agents; thus, efforts to develop strategies for detection and protection are warranted. Fortyeight extracellular proteins of C. perfringens type A and type C strains have been identified here using a 2-dimensional gel electrophoresis-mass spectrometry (2-DE-MS) technique. The SagA protein, the DnaK-type molecular chaperone hsp70, endo-beta-N-acetylglucosaminidase, and hypothetical protein CPF_0656 were among the most abundant proteins secreted by C. perfringens ATCC 13124. The antigenic component of the exoproteome of this strain has also been identified. Most of the extracellular proteins were predicted to be involved in carbohydrate transport and metabolism (16%) or cell envelope biogenesis or to be outer surface protein constituents (13%). More than 50% of the proteins were predictably secreted by either classical or nonclassical pathways. LipoP and TMHMM indicated that nine proteins were extracytoplasmic but cell associated. Immunization with recombinant ornithine carbamoyltransferase (cOTC) clearly resulted in protection against a direct challenge with C. perfringens organisms. A significant rise in IgG titers in response to recombinant cOTC was observed in mice, and IgG2a titers predominated over IgG1 titers (IgG2a/IgG1 ratio, 2). The proliferation of spleen lymphocytes in cOTC-immunized animals suggested a cellular immune response. There were significant increases in the levels of gamma interferon (IFN-␥) and interleukin 2 (IL-2), suggesting a Th1 type immune response.

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