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Genome-wide expression analysis of memory-like inflammatory responses in murine micoglial cells (BV-2 immortalized microglial cell line)

ABSTRACT: Trained immunity and immune tolerance have been identified as long-term response patterns of the innate immune system. The causes of these opposing reactions remain elusive. Here we report about differential inflammatory responses of microglial cells derived from neonatal mouse brain to increasing doses of the endotoxin LPS. Prolonged priming with ultra-low LPS doses provokes trained immunity, i.e. increased production of pro-inflammatory mediators in comparison to the unprimed control. In contrast, priming with high doses of LPS induces immune tolerance implying decreased production of inflammatory mediators and pronounced release of anti-inflammatory cytokines. Investigation of the signaling processes and cell functions involved in these memory-like immune responses reveals essential role of phosphoinositide 3-kinase γ (PI3Kγ), one of the phosphoinositide 3-kinase species highly expressed in innate immune cells. Together, our data suggest profound influence of preceding contacts with pathogens on the immune response of microglia. The impact of these interactions – trained immunity or immune tolerance - appears to be shaped by pathogen dose.

ORGANISM(S): Mus musculus

PROVIDER: GSE137741 | GEO | 2019/12/05

REPOSITORIES: GEO

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Project description:Background: Udder infections with environmental pathogens like Escherichia coli are a serious problem for the diary industry. Reduction of incidence and severity of mastitis is desirable and mild priming of the immune system either through vaccination or with low doses of an immune stimulant like lipopolysaccharide LPS was previously found to dampen detrimental effects of a subsequent infection. Monocytes / macrophages are known to develop tolerance to the endotoxin (ET) LPS as adaptation strategy to prevent exuberant inflammation. We have recently observed that application of 1 M-BM-5g of LPS/udder quarter effectively protects the cow for several days from an experimentally elicited mastitis. We have modelled this process in primary cultures of Mammary Epithelial Cells (MEC) from the cow. This is by far the most abundant cell type in the udder coming into contact with invading pathogens and little is known about the role of MEC in establishing ET in the udder. Results: We primed primary MEC cultures for 6 h with LPS (100 ng/ml) and stimulated some cultures 12 h or 42 h later with 107/ml of E. coli particles. Affymetrix microarrays were used to profile priming related alterations in the global transcriptome of those cells. Corner stone data were validated with quantitative real time PCR. LPS-priming alone caused differential expression of 40 genes and mediated significantly different response to a subsequent E. coli challenge of 226 genes. Expression of 38 genes was enhanced while that of 188 was decreased. Higher expressed were anti-microbial factors (M-CM-^_-defensin LAP, SLPI), cell and tissue protecting factors (DAF, MUC1, TGM1,-3) as well as mediators of the sentinel function of MEC (CCL5, IL-8). Dampened was the expression of potentially harmful pro-inflammatory master cytokines (IL-1B, IL-6, TNF-alpha and immune effectors (iNOS, matrix metalloproteases). Functional network analysis highlighted the reduced expression of IL-1B and of IRF7 as key to this modulation. Conclusion: LPS-primed MEC are fitter to repel pathogens and better protected against misguided attacks of the immune response. Attenuated is the exuberant expression of factors potentially promoting immunopathological processes. MEC therefore recapitulate many aspects of ET known so far from professional immune cells. Each experiment was performed in triplicate using separate pbMEC cultures (80% confluence) prepared from the udders of udders of three healthy, pregnant (d130 of gestation) cows in the mid of their first lactation. Controls (C.) were cultured for 12 h in GM, washed 3x with PBS and cultivated for additional 18 h (short waiting experiment) or 48 h (long waiting experiment). For the priming experiment (P.) the pbMEC cultures were stimulated (M-bM-^@M-^\primedM-bM-^@M-^]) at time 0 h with 100 ng/ml LPS , washed three times with PBS and cultivated for another 18 h or 48 h. For the induction trial (I.) cells were challenged with 107/ml particles of heat inactivated E. coli1303 for 6 h. The cultures were handled exactly like the control group (C.) prior to the E. coli challenge. For the induction post priming experiment (I.p.P.) cultures were primed for 12 h with LPS, just as described for the priming group (P.). After 3x washing with PBS and waiting for 12 h or 42 h in GM the I.p.P. cultures were also challenged with 107/ml particles of E. coli1303. Cells were harvested at time 30 h (short waiting experiments) or 60 h (long waiting experiments) and total RNA was prepared.

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Genome-wide expression analysis of memory-like inflammatory responses in murine micoglial cells (BV-2 immortalized microglial cell line)

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