Supplementary MaterialsSupplemental material for CD11b-activated Src signal attenuates neuroinflammatory pain by orchestrating inflammatory and anti-inflammatory cytokines in microglia Supplemental_material. Src by PP1 increased inflammation in wild-type microglia cells significantly, but not in CD11b-deficient microglia cells. In vivo, CD11b-deficient mice were more susceptible to chronic constrictive injury-induced allodynia and hyperalgesia with significantly more inflammatory cytokines expression. All these results indicated that the regulatory function of CD11b-Src signal pathway on both inflammatory and anti-inflammatory cytokines in microglia cells is a potential target in neuropathic pain treatment. strong class=”kwd-title” SMN Keywords: Neuropathic pain, neuroinflammation, CD11b, Src, TLR4 Introduction Chronic pain is typically characterized by hyperalgesia, which is an increased response to noxious thermal or mechanical stimuli, as well as allodynia, nociceptive responses which occur to normally innocuous stimuli even light touch (known as mechanical allodynia).1C3 Inflammation is defined as homeostatic interaction between the immune system and MK-8776 cost injury tissues. Inflammatory mediators, such as misfolded proteins, aberrantly localized nucleic acids, reactive oxygen species (ROS), H+, adenosine-triphosphate (ATP) bradykinin, prostaglandins, MK-8776 cost nerve growth factor, and pro-inflammatory cytokines and chemokines, released locally after tissue injury can directly stimulate and cause sensitization of pain-sensing nociceptors located at nerve fibers of primary afferent neurons MK-8776 cost in peripheral tissue.4C8 The innate immune receptors in microglia cells can directly respond to damage-associated molecular patterns (DAMPs) or pathogen-associated molecular patterns (PAMPs).9,10 Abnormal activation of microglia cells leads to the sustained exposure of neurons to pro-inflammatory mediators and causes neuronal dysfunction or cell death. Thus, activation and regulation of microglia cells are critical for neuron system homeostasis. Microglia expressed a board spectrum of pattern-recognition receptors (PRRs), including toll-like receptor (TLR) 2, TLR4, and TLR6. As there is a strong overlap between the signaling pathways induced by PAMPs and DAMPs, microglia may not be able to discriminate invading pathogens and misfolded or aberrant endogenous molecular patterns.11C13 Inappropriate activation of PRRs can lead to prolonged inflammation, which induces both pro-inflammatory cytokines, such as interleukin (IL)-6 and tumor necrosis factor alpha (TNF-), and anti-inflammatory cytokines, such as IL-10 and transforming growth factor beta (TGF-).9,10,14 Although the molecular regulatory mechanism of TLR signaling in macrophages has been under intensive research, it remains unknown whether there is a potential signal pathway orchestrating both inflammatory and anti-inflammatory cytokines. Thus, it is critical to determine the balance of PRRs signal transduction pathway in inflammatory response and immune disorders in order to maintain innate immune response. CD11b, a member of integrin family, is highly expressed on monocytes/macrophages, common dendritic cells (DCs), and microglia cells.15,16 CD11b expressed on DCs inhibited Th17 differentiation and T-cell activation.17,18 Stromal microenvironment of spleen, liver, and lung can drive generation of a new population of CD11bhi regulatory DCs with higher IL-10 production, which could inhibit T-cell activation.19C21 CD11b on macrophages could also inhibit TLR activation-induced inflammatory responses by inhibiting inflammatory cytokines and increasing anti-inflammatory cytokine IL-10 by activating Src-spleen tyrosine kinase (Syk) signaling.22,23 However, the function of integrins on microglia, such as CD11b, remains unknown. Therefore, we made the following assumptions and validate them by experiments: CD11b inhibits inflammatory cytokines IL-6 and TNF- via Src/Syk pathway, and attenuates chronic constrictive injury (CCI)-induced allodynia by promoting TLR-triggered IL-10 expression. Here, we found that CD11b-deficient microglia produced more inflammatory cytokines, such as IL-6 and TNF-, while less anti-inflammatory cytokines, such as IL-10. Signal transduction assay confirmed that CD11b inhibited TLR-signaling and inflammatory cytokines through Src/Syk. CD11b-deficient mice were more susceptible to CCI-induced hypersensitivity. Our results revealed the regulatory function of CD11b-Src signal pathway in orchestrating both inflammatory and anti-inflammatory cytokines in microglia cells, which outlines potential-targeted signal pathway in the treatment of neuropathic pain by Src activator. Materials and methods Animal preparation Male C57 BL/6J mice (20C25 g) were purchased from the Laboratory Animal Center of the Second Military Medical University and caged in groups of four or five. Mice homozygous for Itgam (CD11b) deficiency (B6.129S4-Itgamtm1Myd/J; 003991; Jackson Laboratories, Bar Harbor, ME) were bred in pathogen-free conditions. Mice were maintained 12:12 h light/dark cycle with adequate food and water. All mice were placed in the experimental room 24?h before behavioral test for acclimatization. All experiments were reviewed and approved by the Institutional Animal Care and Use Committee of the Second Military Medical University (Shanghai, China) and conducted in accordance with the American Physiological Societys Guiding Principles in the Treatment and Usage of Animals.24 All male mice had been separated from animal middle randomly. On the entire day time before test, the mice had been randomly designated to either the control group or among the test groups, and test size for every test was approximated by Meads source equation, which work. Primary ethnicities of microglia Microglia had been isolated from.