miR-132 suppresses transcription of ribosomal proteins to promote protective Th1 immunity.

Determining the mechanisms that distinguish protective immunity from pathological chronic inflammation remains a fundamental challenge. miR-132 has been shown to play largely immunoregulatory roles in immunity; however, its role in CD4+ T cell function is poorly understood. Here, we show that CD4+ T cells express high levels of miR-132 and that T cell activation leads to miR-132 up-regulation. The transcriptomic hallmark of splenic CD4+ T cells lacking the miR-132/212 cluster during chronic infection is an increase in mRNA levels of ribosomal protein (RP) genes. BTAF1, a co-factor of B-TFIID and novel miR-132/212-3p target, and p300 contribute towards miR-132/212-mediated regulation of RP transcription. Following infection with Leishmania donovani, miR-132-/- CD4+ T cells display enhanced expression of IL-10 and decreased IFNγ. This is associated with reduced hepatosplenomegaly and enhanced pathogen load. The enhanced IL-10 expression in miR-132-/- Th1 cells is recapitulated in vitro following treatment with phenylephrine, a drug reported to promote ribosome synthesis. Our results uncover that miR-132/212-mediated regulation of RP expression is critical for optimal CD4+ T cell activation and protective immunity against pathogens.

Meningeal Whole Mounts for Imaging CNS Fungal Infection.

Invasive fungal infections may involve the brain and central nervous system (CNS), leading to often fatal meningitis in immunocompromised individuals. Recent technological advances have allowed us to move beyond studying the brain parenchyma to understanding the immune mechanisms of the meninges, the protective layer that surrounds the brain and spinal cord. Specifically, advanced microscopy techniques have enabled researchers to begin to visualize the anatomy of the meninges and the cellular mediators of meningeal inflammation. In this chapter, we describe how to make meningeal tissue mounts for imaging by confocal microscopy.

Microglia are not protective against cryptococcal meningitis.

Microglia provide protection against a range of brain infections including bacteria, viruses and parasites, but how these glial cells respond to fungal brain infections is poorly understood. We investigated the role of microglia in the context of cryptococcal meningitis, the most common cause of fungal meningitis in humans. Using a series of transgenic- and chemical-based microglia depletion methods we found that, contrary to their protective role during other infections, loss of microglia did not affect control of Cryptococcus neoformans brain infection which was replicated with several fungal strains. At early time points post-infection, we found that microglia depletion lowered fungal brain burdens, which was related to intracellular residence of C. neoformans within microglia. Further examination of extracellular and intracellular fungal populations revealed that C. neoformans residing in microglia were protected from copper starvation, whereas extracellular yeast upregulated copper transporter CTR4. However, the degree of copper starvation did not equate to fungal survival or abundance of metals within different intracellular niches. Taken together, these data show how tissue-resident myeloid cells may influence fungal phenotype in the brain but do not provide protection against this infection, and instead may act as an early infection reservoir.

The glucocorticoid dexamethasone inhibits HIF-1α stabilization and metabolic reprogramming in lipopolysaccharide-stimulated primary macrophages.

Synthetic glucocorticoids are used to treat many chronic and acute inflammatory conditions. Frequent adverse effects of prolonged exposure to glucocorticoids include disturbances of glucose homeostasis caused by changes in glucose traffic and metabolism in muscle, liver, and adipose tissues. Macrophages are important targets for the anti-inflammatory actions of glucocorticoids. These cells rely on aerobic glycolysis to support various pro-inflammatory and antimicrobial functions. Employing a potent pro-inflammatory stimulus in two commonly used model systems (mouse bone marrow-derived and human monocyte-derived macrophages), we showed that the synthetic glucocorticoid dexamethasone inhibited lipopolysaccharide-mediated activation of the hypoxia-inducible transcription factor HIF-1α, a critical driver of glycolysis. In both cell types, dexamethasone-mediated inhibition of HIF-1α reduced the expression of the glucose transporter GLUT1, which imports glucose to fuel aerobic glycolysis. Aside from this conserved response, other metabolic effects of lipopolysaccharide and dexamethasone differed between human and mouse macrophages. These findings suggest that glucocorticoids exert anti-inflammatory effects by impairing HIF-1α-dependent glucose uptake in activated macrophages. Furthermore, harmful and beneficial (anti-inflammatory) effects of glucocorticoids may have a shared mechanistic basis, depending on the alteration of glucose utilization.