TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ

Abstract

Microglia (tissue-resident macrophages) represent the main cell type of the innate immune system in the CNS; however, the mechanisms that control the activation of microglia are widely unknown. We systematically explored microglial activation and functional microglia-neuron interactions in organotypic hippocampal slice cultures, i.e., postnatal cortical tissue that lacks adaptive immunity. We applied electrophysiological recordings of local field potential and extracellular K(+) concentration, immunohistochemistry, design-based stereology, morphometry, Sholl analysis, and biochemical analyses. We show that chronic activation with either bacterial lipopolysaccharide through Toll-like receptor 4 (TLR4) or leukocyte cytokine IFN-γ induces reactive phenotypes in microglia associated with morphological changes, population expansion, CD11b and CD68 up-regulation, and proinflammatory cytokine (IL-1β, TNF-α, IL-6) and nitric oxide (NO) release. Notably, these reactive phenotypes only moderately alter intrinsic neuronal excitability and gamma oscillations (30-100 Hz), which emerge from precise synaptic communication of glutamatergic pyramidal cells and fast-spiking, parvalbumin-positive GABAergic interneurons, in local hippocampal networks. Short-term synaptic plasticity and extracellular potassium homeostasis during neural excitation, also reflecting astrocyte function, are unaffected. In contrast, the coactivation of TLR4 and IFN-γ receptors results in neuronal dysfunction and death, caused mainly by enhanced microglial inducible nitric oxide synthase (iNOS) expression and NO release, because iNOS inhibition is neuroprotective. Thus, activation of TLR4 in microglia in situ requires concomitant IFN-γ receptor signaling from peripheral immune cells, such as T helper type 1 and natural killer cells, to unleash neurotoxicity and inflammation-induced neurodegeneration. Our findings provide crucial mechanistic insight into the complex process of microglia activation, with relevance to several neurologic and psychiatric disorders.

Keywords: Toll-like receptor; hippocampus; microglia; neuronal activity; slice culture.

Fig. 1.

Cytokine release. Slice cultures were exposed to LPS (10 µg/mL), IFN-γ (100 ng/mL), or both (LPS+IFN-γ) at day in vitro 7 (SI Appendix, Fig. S1). (A–D) (Left) Profile of cytokine accumulation in the medium, normalized to the number of slice cultures per membrane. Dots and error bars represent mean ± SEM. *P < 0.001 of LPS and LPS+IFN-γ vs. CTL and IFN-γ, one-way ANOVA or ANOVA on ranks. For n/N membranes/preparations: CTL, 15/7; LPS, 19/7; IFN-γ, 21/7; LPS+IFN-γ, 19/7. (Right) Histograms of cytokine levels after 72 h of exposure. Boxplots: *P < 0.05, ANOVA on ranks vs. CTL; ⁺P < 0.05, rank-sum test. CTL, 37/14; LPS, 34/12; IFN-γ, 33/11; LPS+IFN-γ, 37/12. IFN-γ levels most likely derive from the added protein. Control experiments are shown in SI Appendix, Fig. S2.

Fig. 2.

Morphological correlates of microglial activation as revealed by Iba1 immunohistochemistry. (A) CA3 region of control (CTL) and slice cultures exposed to LPS (10 µg/mL), IFN-γ (100 ng/mL), or NMDA+KA (5 µM) for 48 h to induce excitotoxic death. (B) Design-based stereology in cryosections (25 µm) of individual slice cultures. The dentate gyrus is in yellow, and CA3-CA1 is in blue. (Inset) Interpolated 3D reconstruction of sampling contours with randomly sampled microglial cells (dots). Estimates refer to the total hippocampus. (C) Stereological estimation of Iba1-positive cells per slice culture with the optical fractionator probe. *P < 0.05 vs. control, ANOVA on ranks. n/N as in D. (D) Stereological estimation of total process length per cell with the spaceballs probe. *P < 0.05 vs. control, one-way ANOVA. For n/N cultures/preparations: CTL, 10/5; LPS, 13/5; IFN-γ, 9/4; NMDA+KA, 10/4. (E) Parameterization of 2D somatic projections based on maximum length (L) and projection area (A). The somatic shape index (L/A) increases in rod-shaped somata. Images, spectrum of microglial shapes. (F–H) Microglial somatic L, A, and L/A. *P < 0.05, rank-sum test. CTL, 114 cells/9/7; LPS, 102 cells/10/5. (I) Three-dimensional Sholl grid of concentric spheres describing microglial branch length and number as a function of distance from the geometric center of the cell somata (red cross). Branch intersections with the spherical grid quantify the number, whereas the branch length between consecutive spheres estimates the degree of convolution. (J and K) Sholl analysis of microglial processes. IS, intersections. CTL, 116 cells/9/7; LPS, 102 cells/10/5. (L) Fluorescent images of ramified and ameboid microglia. (M) Stereological quantification of ameboid cells with enlarged, round somata (cell bodies) showing no more than a single process and a few filopodia, with the optical fractionator probe. P > 0.05, rank-sum test. CTL, 11/11; LPS, 7/7.

Fig. 3.

iNOS-mediated neurodegeneration. (A) Immunohistochemistry of MAP2 (Left), parvalbumin in CA3 (Middle), and iNOS in cryosections (25 µm) (Right). Note the complex morphology of parvalbumin-positive GABAergic interneurons (fine meshwork, black) contacting the perisomatic region of pyramidal cells. (B) Cell death revealed by LDH activity in the medium after 72 h of exposure. For n/N membranes/preparations: CTL, 36/11; LPS, 34/12; IFN-γ, 33/11; LPS+IFN-γ, 37/12; NMDA+KA, 21/7. (C) Griess reaction for nitrite to detect NO release. For n/N membranes/preparations: CTL, 8/3; LPS, 8/3; IFN-γ, 8/3; LPS+IFN-γ, 8/3; 1400W, 9/3; 1400W+LPS+IFN-γ, 9/3. *P < 0.05 vs. control, ANOVA on ranks with Dunn´s post hoc pairwise test; ⁺P < 0.001, rank-sum or t test (B and C). (D) iNOS staining in stratum pyramidale of CA3.

Fig. 4.

fEPSP kinetics, short-term synaptic plasticity, and K⁺ homeostasis. (A) fEPSP responses in CA3 plotted as normalized to their maximum amplitude to better appreciate the fEPSP kinetics. Solid lines represent grand averages of normalized fEPSP traces ± 1σ (dotted lines). (B) The time to peak between electrical stimulation and maximum fEPSP amplitude, describing the fEPSP kinetics. It is slower for IFN-γ and 1400W+LPS+IFN-γ, but unchanged when comparing 1400W and 1400W+LPS+IFN-γ. For n/N cultures/preparations: CTL, 78/9; LPS, 65/7; IFN-γ, 48/6; 1400W, 12/2; 1400W+LPS+IFN-γ, 80/3. *P < 0.05, ANOVA on ranks with Dunn´s post hoc pairwise test. n.s., nonsignificant. (C) fEPSP responses in CA3 evoked by paired-pulse stimulation at 50% of maximum response (stimulation artifacts truncated). Grand averages of paired-pulse fEPSP responses with an interstimulus interval (ISI) of 100 ms, normalized to the maximum amplitude as evoked by the first pulse. P1, first pulse; P2, second pulse. The paired-pulse index (PPI) is defined as the ratio P2/P1. (D and E) The PPI of 1400W+LPS+IFN-γ and 1400W is significantly lower compared with that of CTL, LPS, and IFN-γ for all ISIs. P < 0.05, ANOVA on ranks. There are no differences among CTL, LPS, and IFN-γ, or between 1400W+LPS+IFN-γ and 1400W. For n/N cultures/preparations: CTL, 23/9; LPS, 12/7; IFN-γ, 15/6; 1400W, 3/2; 1400W+LPS+IFN-γ, 14/3. P > 0.05, ANOVA on ranks. (F) Extracellular K⁺ homeostasis during electrical stimulation (5 s, 20 Hz, 1.5 V; black bar) of fiber tracts projecting to CA1. The double-barreled microelectrode ([K⁺]_o and LFP) was positioned in stratum pyramidale of CA1. Increase, decay, and undershoot of [K⁺]_o transients reflect K⁺ release from neurons, K⁺ (re)uptake from neurons and astrocytes, and K⁺ redistribution within the gap junction-coupled astrocytic syncytium, respectively. The traces illustrate average responses for control (black) and LPS exposure (10 µg/mL, for 72 h) (green). (G) Increase and undershoot of stimulus-induced [K⁺]_o transients were similar for control and LPS. P > 0.05, rank-sum test. For n/N cultures/preparations: CTL, 15/10; LPS, 22/10. (H) Original LFP trace (gray) during stimulation (5 s, 20 Hz, 1.5 V; black bar) with filtered slow negativity (black dotted line). (I) Average slow DC-negativity during stimulation (5 s, 20 Hz). Gray area indicates period with significant differences. *P < 0.05, rank-sum test. n/N as in G.

Fig. 5.

Fast network oscillations. (A) Gamma oscillations in stratum pyramidale of CA3 in the presence of acetylcholine (2 µM) and physostigmine (400 nM). LFP traces (Left) with corresponding power spectrograms for 10–130 Hz (Right). (B) Peak power spectral density derived from power spectrograms (A, Right). *P < 0.05, ANOVA on ranks. n.s., nonsignificant. (C) Peak frequency derived from power spectrograms (A, Right). *P < 0.05, ANOVA on ranks; +P < 0.05, rank-sum test. For n/N cultures/preparations: CTL, 12/5; LPS, 9/3; IFN-γ, 8/3; 1400W, 40/5; 1400W+LPS+IFN-γ, 33/5; LPS+IFN-γ, 10/3.