Elucidating the basis of brain control of immune responses

National Institute Of Allergy And Infectious Diseases

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Abstract

The CSF is produced by the choroid plexus and circulates through the ventricular system before reaching subarachnoid space. We hypothesized brain regions adjacent to CSF-filled ventricles actively modify CSF composition, thereby shaping brain-wide immunity. We systemically interrogated ventricle-contacting brain nuclei for molecular signatures indicative of potential functions in immune sensing and defense functions. Using single-cell RNA (scRNA) sequencing, we identified a specialized population of GABAergic inhibitory neurons in the caudal nucleus of the solitary tract (cNST) that express several members of the Defa (defensin alpha) gene family. Defensins are a large array of evolutionary conserved, cysteine-rich cationic proteins known for their potent antimicrobial activity. These Defa+ neurons abut area postrema, one of the sensory circumventricular organs (CVO) harboring fenestrated blood vessels, and 4th ventricle, thus poised to sample blood-derived signals and access the CSF content. We modeled bacterial meningitis in mice by intravenously injecting a clinically relevant pathogenic strain E.coli (Escherichia coli) K1. Infection led to activation of Defa⁺ neurons in the cNST. Upon activation, these neurons upregulated Defa expression in the brainstem and released DEFA protein into the CSF, akin to canonical immune cells enhancing their effector functions in response to infections. We will examine whether a similar defensin expressing population exists in human brain by staining human postmortem brain tissue. If identified, we will further determine whether neuron-derived defensins are elevated in the CSF of meningitis patients. Together, these results uncovered a new inhibitory neuronal population that is loaded with antimicrobial peptides and strategically positioned at the interface of periphery-CSF communication, exhibiting characteristics of brain immune sentinels. Next we explored whether Defa+ neurons protect the brain from bacterial infections. To access and manipulate these neurons, we developed a Cre knock-in line that reliably marks Defa+ neurons. Genetic ablation of Defa+ neurons led to an increased E.coli K1 load in the brain without affecting bacterial levels in peripheral organs of the spleen or blood, demonstrating that these neurons confer essential immunity against bacterial infections. Chemogenetic silencing of these neurons similarly increased bacterial burdens in the brain, substantiating the importance of neural activity in driving effective antibacterial responses. Selective depletion of DEFA from cNST neurons resulted in uncontrolled E.coli K1 brain infection, highlighting neuron-derived defensins as key effector molecules in containing E.coli infection. Together, these experiments exposed Defa+ neurons and alpha defensins as new “warrior” and “arsenal” in brain antibacterial immunity. We will expand our study to test whether this neuronal defensin system provides similar protection against other categories of meningitis-causing pathogens such as gram positive bacterium streptococcus agalactiae and viral agents of lymphocytic choriomeningitis virus (LCMV) and herpes simplex virus (HSV). How do neuron-derived defensins control brain bacterial infection? Some defensins exhibit bactericidal activity against certain pathogens. Treating E.coli K1 with these defensins did not induce bacterial death even at the exceedingly high DEFA concentration, indicating the presence of alternative protective mechanisms beyond direct bacterial killing. We therefore hypothesized that Defa+ neurons regulate brain immune cell responses. Chemogenetic activation of Defa+ neurons enhanced brain leukocyte number in mice infected with E.coli K1, with heightened recruitment of myeloid (macrophages, neutrophils and monocytes) and lymphoid (CD8+ and CD4+ T lymphocytes) cells. Conversely, silencing of these neurons chemogentically inhibited leukocyte infiltration into the brain following K1 infection. Remarkably, activation of Defa+ neurons alone or delivering DEFA into the CSF in the absence of any infection was sufficient to drive the expansion of inflammatory immune cells in the brain, highlighting the extraordinary capacity of these neurons to activate inflammatory responses to combat bacterial infections. We will further combine sc-RNA seq, mouse genetics and in vitro models to elucidate cellular and molecular mechanisms enabling the immune activation by DEFA – which immune cells are primarily targeted by DEFA, and is there a known receptor mediating its effect?