Systems-Level Proteomics Evaluation of Microglia Response to Tumor-Supportive Anti-Inflammatory Cytokines

Abstract

BackgroundMicroglia safeguard the CNS against injuries and pathogens, and in the presence of certain harmful stimuli are capable of inducing a disease-dependent inflammatory response. When exposed to anti-inflammatory cytokines, however, these cells possess the ability to switch from an inflammatory to an immunosuppressive phenotype. Cancer cells exploit this property to evade the immune system, and elicit an anti-inflammatory microenvironment that facilitates tumor attachment and growth.ObjectiveThe tumor-supportive biological processes that are activated in microglia cells in response to anti-inflammatory cytokines released from cancer cells were explored with mass spectrometry and proteomic technologies.MethodsSerum-depleted and non-depleted human microglia cells (HMC3) were treated with a cocktail of IL-4, IL-13, IL-10, TGFβ, and CCL2. The cellular protein extracts were analyzed by LC-MS/MS. Using functional annotation clustering tools, statistically significant proteins that displayed a change in abundance between cytokine-treated and non-treated cells were mapped to their biological networks and pathways.ResultsThe proteomic analysis of HMC3 cells enabled the identification of ~10,000 proteins. Stimulation with anti-inflammatory cytokines resulted in the activation of distinct, yet integrated clusters of proteins that trigger downstream a number of tumor-promoting biological processes. The observed changes could be classified into four major categories, i.e., mitochondrial gene expression, ECM remodeling, immune response, and impaired cell cycle progression. Intracellular immune activation was mediated mainly by the transducers of MAPK, STAT, TGFβ, NFKB, and integrin signaling pathways. Abundant collagen formation along with the expression of additional receptors, matrix components, growth factors, proteases and protease inhibitors, was indicative of ECM remodeling processes supportive of cell-cell and cell-matrix adhesion. Overexpression of integrins and their modulators was reflective of signaling processes that link ECM reorganization with cytoskeletal re-arrangements supportive of cell migration. Antigen processing/presentation was represented by HLA class I histocompatibility antigens, and correlated with upregulated proteasomal subunits, vesicular/viral transport, and secretory processes. Immunosuppressive and proangiogenic chemokines, as well as anti-angiogenic factors, were detectable in low abundance. Pronounced pro-inflammatory, chemotactic or phagocytic trends were not observed, however, the expression of certain receptors, signaling and ECM proteins indicated the presence of such capabilities.ConclusionsComprehensive proteomic profiling of HMC3 cells stimulated with anti-inflammatory cytokines revealed a spectrum of microglia phenotypes supportive of cancer development in the brain via microenvironment-dependent biological mechanisms.

Persistent DNA damage associated with ATM kinase deficiency promotes microglial dysfunction

“…We observed TNFα-dependent: (i) phosphorylation of p65 at S536 indicative of increased IKK activity ( 50 ) and relocalization of p65 in the nucleus (Figure 2A and B ; Supplementary Figure S5A–D ), (ii) processing of p105 to p50 as evidenced by an increase in p50/p105 ratio and nuclear translocation of p50 (Figure 2A and B ; Supplementary Figures S5A and B ) and (iii) degradation of the NF-κB-inhibitory protein IκBα that was rescued upon treatment with the proteasomal inhibitor MG-132 (Figures 2B and 2C ; Supplementary Figure 5E ) in both WT and ATM KO microglia. Although we did not observe relocalization of c-REL following TNFα treatment (Figure 2B ), its nuclear presence was detected upon treatment of HMC3 microglia with a cocktail of anti-inflammatory cytokines, indicating its context-dependent functions ( 51 ). These data indicate that WT and ATM KO HMC3 microglia are proficient in TNFα-induced canonical NF-κB signalling.…”

Protective Signature of IFNγ-Stimulated Microglia Relies on miR-124-3p Regulation From the Secretome Released by Mutant APP Swedish Neuronal Cells

“…Remarkably, miR-124-3p was confirmed as the miRNA better explaining the full proteomic changes detected in IFNγ-MG, through the predicted involvement of several transcription factors, such as C/EBPα, NFATc1, or STAT3, which also constitute major miR-124 targets. Our coculture system was more effective in the identification of relevant microglial proteomic data compared to other studies using monoculture ( Ahuja and Lazar, 2021 ), despite the differences to more advanced experimental models of microglia ( Svoboda et al, 2019 ) ( Supplementary Figure S4 ). Nevertheless, the sum of these data elucidates the multi-targeting power of neuronal miR-124 over the microglial proteome.…”

Stimuli‐Responsive Miktoarm Polymer‐Based Formulations for Fisetin Delivery and Regulatory Effects in Hyperactive Human Microglia

“…[ 123 ] In contrast, the activation of NFκB by LPS is mitigated in HMC3 microglia in the absence of serum. [ 117 ] Free and nanoformulated fisetin effectively reduced LPS‐induced ROS formation as well as phosphorylation of ERK1/2 (Figure 6B,C). Stimuli‐responsive constructs (M1 and M3) seemed more effective than the control construct (M2) during short‐term treatments (1 h), as fisetin release differs at early time points (Figures 4B,C and 6B).…”

“…We chose human microglia HMC3 cells authenticated by short‐tandem repeat profiling at the American Type Culture Collection (ATCC), and conducted the main experiments in serum‐depleted media. [ 117 ] We opted for these conditions because they provided more clearly biological and immunological functions of HMC3 human microglia upon treatment with pro‐ and anti‐inflammatory agents than in serum‐containing media. [ 117 ] In addition, under physiological conditions, the brain does not contain serum.…”

“…[ 117 ] We opted for these conditions because they provided more clearly biological and immunological functions of HMC3 human microglia upon treatment with pro‐ and anti‐inflammatory agents than in serum‐containing media. [ 117 ] In addition, under physiological conditions, the brain does not contain serum.…”