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 the 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.

Recent studies suggest a role for autophagy in the secretion of IL-1 cytokines regulating the development of inflammatory diseases. The antimalarial drug and autophagy/lysosome inhibitor chloroquine (CHQ) is considered as potential trigger of drug-induced or drug-aggravated psoriasis, in which Th17 cells sustain a persistent inflammation. In this study, we investigated the effect of CHQ on human monocyte-derived Langerhans-like cells (MoLC) and dendritic cells (MoDC) in response to IL-1β. The presence of CHQ reduced IL-12p70 release in both subsets, but surprisingly increased IL-6 production in MoDC and IL-23 in MoLC. Importantly, CHQ-treated MoLC promoted IL-17A secretion by CD4+ T cells and elevated RORC mRNA levels, whereas IFN-γ release was reduced. The dysregulation of IL-12 family cytokines in MoLC and MoDC occurred at the transcriptional level. Similar effects were obtained with other late autophagy inhibitors, whereas PI3K inhibitor 3-methyladenine failed to increase IL-23 secretion. The modulated cytokine release was dependent on IL-1 cytokine activation and abrogated by a specific IL-1R antagonist. CHQ elevated expression of TNFR-associated factor 6, a common intermediate in IL-1R and TLR-dependent signaling. Accordingly, treatment with Pam3CSK4 and CHQ enhanced IL-23 release in MoLC and MoDC. CHQ inhibited autophagic flux, confirmed by increased LC3-II and p62 expression, and activated ERK, p38, and JNK MAPK, but only inhibition of p38 abrogated IL-23 release by MoLC. Thus, our findings indicate that CHQ modulates cytokine release in a p38-dependent manner, suggesting an essential role of Langerhans cells and dendritic cells in CHQ-provoked psoriasis, possibly by promoting Th17 immunity.

A growing body of evidence suggests that innate immune cells can respond in a memory-like (adaptive) fashion, which is referred to as trained immunity. Only few in vivo studies have shown training effects in neutrophils; however, no in vitro setup has been established to study the induction of trained immunity or tolerance in neutrophils by microbial agents. In light of their short lifespan (up to 48 h), we suggest to use the term trained sensitivity for neutrophils in an in vitro setting. Here, we firstly describe a feasible two-hit model, using different doses of lipopolysaccharide (LPS) in bone marrow neutrophils. We found that low doses (10 pg/mL) induce pro-inflammatory activation (trained sensitivity), whereas priming with high doses (100 ng/mL) leads to suppression of pro-inflammatory mediators such as TNF-α or IL-6 (tolerance) (p < 0.05). On a functional level, trained neutrophils displayed increased phagocytic activity and LFA-1 expression as well as migrational capacity and CD11a expression, whereas tolerant neutrophils show contrasting effects in vitro. Mechanistically, TLR4/MyD88/PI3Ks regulate the activation of p65, which controls memory-like responses in mouse bone marrow neutrophils (p < 0.05). Our results open a new window for further in vitro studies on memory-like inflammatory responses of short-lived innate immune cells such as neutrophils.

Microglia, the innate immune cells of the central nervous system, feature adaptive immune memory with implications for brain homeostasis and pathologies. However, factors involved in the emergence and regulation of these opposing responses in microglia have not been fully addressed. Recently, we showed that microglia from the newborn brain display features of trained immunity and immune tolerance after repeated contact with pathogens in a dose-dependent manner. Here, we evaluate the impact of developmental stage on adaptive immune responses of brain microglia after repeated challenge with ultra-low (1 fg/ml) and high (100 ng/ml) doses of the endotoxin LPS in vitro. We find that priming of naïve microglia derived from newborn but not mature and aged murine brain with ultra-low LPS significantly increased levels of pro-inflammatory mediators TNF-α, IL-6, IL-1β, MMP-9, and iNOS as well as neurotrophic factors indicating induction of trained immunity (p < 0.05). In contrast, stimulation with high doses of LPS led to a robust downregulation of pro-inflammatory cytokines and iNOS independent of the developmental state, indicating induced immune tolerance. Furthermore, high-dose priming with LPS upregulated anti-inflammatory mediators IL-10, Arg-1, TGFβ, MSR1, and IL-4 in newborn microglia (p < 0.05). Our data indicate pronounced plasticity of the immune response of neonate microglia compared with microglia derived from mature and aged mouse brain. Induced trained immunity after priming with ultra-low LPS doses may be responsible for enhanced neuro-inflammatory susceptibility of immature brain. In contrast, the immunosuppressed phenotype following high-dose LPS priming might be prone to attenuate excessive damage after recurrent systemic inflammation.

Newborns are highly susceptible to infections and mainly rely on innate immune functions. Reduced reactivity, delayed activation and subsequent failure to resolve inflammation however makes the neonatal immune system a very volatile line of defense. Perinatal microbiota, nutrition and different extra-uterine factors are critical elements that define long-term outcomes and shape the immune system during the neonatal period. Neutrophils are first responders and represent a vital component of the immune system in newborns. They have long been regarded as merely executive immune cells, however this notion is beginning to shift. Neutrophils are shaped by their surrounding and adaptive elements have been described. The role of “innate immune memory” and the main triangle connection microbiome—neutrophil—adaptation will be discussed in this review.

Microglia, the innate immune cells of the CNS, exhibit long-term response changes indicative of innate immune memory (IIM). Our previous studies revealed IIM patterns of microglia with opposing immune phenotypes: trained immunity after a low dose and immune tolerance after a high dose challenge with pathogen-associated molecular patterns (PAMP). Compelling evidence shows that innate immune cells adopt features of IIM via immunometabolic control. However, immunometabolic reprogramming involved in the regulation of IIM in microglia has not been fully addressed. Here, we evaluated the impact of dose-dependent microglial priming with ultra-low (ULP, 1 fg/mL) and high (HP, 100 ng/mL) lipopolysaccharide (LPS) doses on immunometabolic rewiring. Furthermore, we addressed the role of PI3Kγ on immunometabolic control using naïve primary microglia derived from newborn wild-type mice, PI3Kγ-deficient mice and mice carrying a targeted mutation causing loss of lipid kinase activity. We found that ULP-induced IIM triggered an enhancement of oxygen consumption and ATP production. In contrast, HP was followed by suppressed oxygen consumption and glycolytic activity indicative of immune tolerance. PI3Kγ inhibited glycolysis due to modulation of cAMP-dependent pathways. However, no impact of specific PI3Kγ signaling on immunometabolic rewiring due to dose-dependent LPS priming was detected. In conclusion, immunometabolic reprogramming of microglia is involved in IIM in a dose-dependent manner via the glycolytic pathway, oxygen consumption and ATP production: ULP (ultra-low-dose priming) increases it, while HP reduces it.

Neutrophils are classically characterized as merely reactive innate effector cells. However, the microbiome is known to shape the education and maturation process of neutrophils, improving their function and immune-plasticity. Recent reports demonstrate that murine neutrophils possess the ability to exert adaptive responses after exposure to bacterial components such as LPS (Gram-negative bacteria) or LTA (Gram-positive bacteria). We now ask whether small extracellular vesicles (EVs) from the gut may directly mediate adaptive responses in neutrophils in vitro. Murine bone marrow-derived neutrophils were primed in vitro by small EVs of high purity collected from colon stool samples, followed by a second hit with LPS. We found that low-dose priming with gut microbiota-derived small EVs enhanced pro-inflammatory sensitivity as indicated by elevated levels of TNF-α, IL-6, ROS and MCP-1 and increased migratory and phagocytic activity. In contrast, high-dose priming resulted in a tolerant phenotype, marked by increased IL-10 and decreased transmigration and phagocytosis. Alterations in TLR2/MyD88 as well as TLR4/MyD88 signaling were correlated with the induction of adaptive cues in neutrophils in vitro. Taken together, our study shows that small EVs from stools can drive adaptive responses in neutrophils in vitro and may represent a missing link in the gut–immune axis.

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