CD40-CD154: A perspective from type 2 immunity.

The interaction between CD40 and CD154 (CD40 ligand) is central in immunology, participating in CD4+ T cell priming by dendritic cells (DC), CD4+ T cell help to B cells and classical macrophage activation by CD4+ T cells. However, its role in the Th2 side of immunology including helminth infection remains incompletely understood. Contrary to viral and bacterial stimuli, helminth products usually do not cause CD40 up-regulation in DC, and exogenous CD40 ligation drives Th2-biased systems towards Th1. On the other hand, CD40 and CD154 are necessary for induction of most Th2 responses. We attempt to reconcile these observations, mainly by proposing that (i) CD40 up-regulation in DC in Th2 systems is mostly induced by alarmins, (ii) the Th2 to Th1 shift induced by exogenous CD40 ligation is related to the capacity of such ligation to enhance IL-12 production by myeloid cells, and (iii) signals elicited by endogenous CD154 available in Th2 contexts and by exogenous CD40 ligation are probably different. We stress that CD40-CD154 is important beyond cognate cellular interactions. In such a context, we argue that the proliferation response of B-cells to IL-4 plus CD154 reflects a Th2-specific mechanism for polyclonal B-cell amplification and IgE production at infection sites. Finally, we argue that CD154 is a general immune activation signal across immune polarization including Th2, and propose that competition for CD154 at tissue sites may provide negative feedback on response induction at each site.

SIRT6 stabilization and cytoplasmic localization in macrophages regulates acute and chronic inflammation in mice.

Acute and chronic inflammations are key homeostatic events in health and disease. Sirtuins (SIRTs), a family of NAD-dependent protein deacylases, play a pivotal role in the regulation of these inflammatory responses. Indeed, SIRTs have anti-inflammatory effects through a myriad of signaling cascades, including histone deacetylation and gene silencing, p65/RelA deacetylation and inactivation, and nucleotide­binding oligomerization domain, leucine rich repeat, and pyrin domain­containing protein 3 inflammasome inhibition. Nevertheless, recent findings show that SIRTs, specifically SIRT6, are also necessary for mounting an active inflammatory response in macrophages. SIRT6 has been shown to positively regulate tumor necrosis factor alpha (TNFα) secretion by demyristoylating pro-TNFα in the cytoplasm. However, how SIRT6, a nuclear chromatin-binding protein, fulfills this function in the cytoplasm is currently unknown. Herein, we show by Western blot and immunofluorescence that in macrophages and fibroblasts there is a subpopulation of SIRT6 that is highly unstable and quickly degraded via the proteasome. Upon lipopolysaccharide stimulation in Raw 264.7, bone marrow, and peritoneal macrophages, this population of SIRT6 is rapidly stabilized and localizes in the cytoplasm, specifically in the vicinity of the endoplasmic reticulum, promoting TNFα secretion. Furthermore, we also found that acute SIRT6 inhibition dampens TNFα secretion both in vitro and in vivo, decreasing lipopolysaccharide-induced septic shock. Finally, we tested SIRT6 relevance in systemic inflammation using an obesity-induced chronic inflammatory in vivo model, where TNFα plays a key role, and we show that short-term genetic deletion of SIRT6 in macrophages of obese mice ameliorated systemic inflammation and hyperglycemia, suggesting that SIRT6 plays an active role in inflammation-mediated glucose intolerance during obesity.

CD300f immune receptor contributes to healthy aging by regulating inflammaging, metabolism, and cognitive decline.

Emerging evidence suggests that immune receptors may participate in many aging-related processes such as energy metabolism, inflammation, and cognitive decline. CD300f, a TREM2-like lipid-sensing immune receptor, is an exceptional receptor as it integrates activating and inhibitory cell-signaling pathways that modulate inflammation, efferocytosis, and microglial metabolic fitness. We hypothesize that CD300f can regulate systemic aging-related processes and ultimately healthy lifespan. We closely followed several cohorts of two strains of CD300f-/- and WT mice of both sexes for 30 months and observed an important reduction in lifespan and healthspan in knockout mice. This was associated with systemic inflammaging, increased cognitive decline, reduced brain glucose uptake observed by 18FDG PET scans, enrichment in microglial aging/neurodegeneration phenotypes, proteostasis alterations, senescence, increased frailty, and sex-dependent systemic metabolic changes. Moreover, the absence of CD300f altered macrophage immunometabolic phenotype. Taken together, we provide strong evidence suggesting that myeloid cell CD300f immune receptor contributes to healthy aging.

Flow Cytometry Analysis of SIRT6 Expression in Peritoneal Macrophages.

The sirtuin 6 has emerged as a regulator of acute and chronic immune responses. Recent findings show that SIRT6 is necessary for mounting an active inflammatory response in macrophages. In vitro studies revealed that SIRT6 is stabilized in the cytoplasm to promote tumor necrosis factor (TNFα) secretion. Notably, SIRT6 also promotes TNFα secretion by resident peritoneal macrophages upon lipopolysaccharide (LPS) stimulation in vivo. Although many studies have investigated SIRT6 function in the immune response through different genetic and pharmacological approaches, direct measurements of in vivo SIRT6 expression in immune cells by flow cytometry have not yet been performed. Here, we describe a step-by-step protocol for peritoneal fluid extraction, isolation, and preparation of peritoneal cavity cells, intracellular SIRT6 staining, and flow cytometry analysis to measure SIRT6 levels in mice peritoneal macrophages. By providing a robust method to quantify SIRT6 levels in different populations of macrophages, this method will contribute to deepening our understanding of the role of SIRT6 in immunity, as well as in other cellular processes regulated by SIRT6. Graphical abstract.