AMPK induces regulatory innate lymphoid cells after traumatic brain injury

Abstract

The CNS is regarded as an immunoprivileged organ, evading routine immune surveillance; however, the coordinated development of immune responses profoundly influences outcomes after brain injury. Innate lymphoid cells (ILCs) are cytokine-producing cells that are critical for the initiation, modulation, and resolution of inflammation, but the functional relevance and mechanistic regulation of ILCs are unexplored after acute brain injury. We demonstrate increased proliferation of all ILC subtypes within the meninges for up to 1 year after experimental traumatic brain injury (TBI) while ILCs were present within resected dura and elevated within cerebrospinal fluid (CSF) of moderate-to-severe TBI patients. In line with energetic derangements after TBI, inhibition of the metabolic regulator, AMPK, increased meningeal ILC expansion, whereas AMPK activation suppressed proinflammatory ILC1/ILC3 and increased the frequency of IL-10-expressing ILC2 after TBI. Moreover, intracisternal administration of IL-33 activated AMPK, expanded ILC2, and suppressed ILC1 and ILC3 within the meninges of WT and Rag1-/- mice, but not Rag1-/- IL2rg-/- mice. Taken together, we identify AMPK as a brake on the expansion of proinflammatory, CNS-resident ILCs after brain injury. These findings establish a mechanistic framework whereby immunometabolic modulation of ILCs may direct the specificity, timing, and magnitude of cerebral immunity.

Keywords: Immunology; Neurological disorders; Neuroscience.

Figure 1. Presence and frequency of ILC subtypes within the meninges of severe TBI patients.

(A) Dura was collected from consecutive, severe TBI patients undergoing decompressive craniectomy to alleviate elevated intracranial pressure. ILCs were sorted using forward scatter (FSC)/side scatter (SSC) and identified as CD45⁺, lineage-negative (Lin⁻), CD127⁺ lymphoid cells. ILCs subtypes were further defined as ILC1:, CD45⁺Lin⁻CD127⁺CD161⁺NKp44⁺; ILC2, CD45⁺Lin⁻CD127⁺GATA3⁺CRTH2⁺; and ILC3, CD45⁺Lin⁻CD127⁺RORγt⁺AhR⁺, as shown in representative flow cytometry scatterplots. Gray shaded areas indicate isotype controls. To demonstrate functionality, ILCs were further stimulated with cytokine cocktails, and production of signature cytokines was assessed (ILC1, IFN-γ; ILC2, IL-5/IL-13; ILC3, IL-17). (B and C) Frequency of ILC subtypes from individual patients, expressed as total cell number (B) and % leukocytes (C) (n = 5). Scatterplots depict mean ± SD. (D) Computed tomography scan of a TBI patient before (Pre-) and after (Post-) decompressive craniectomy surgery. The dura was collected during surgery at the time of bone flap removal.

Figure 2. Increased presence of ILC1 and ILC3 within human CSF after TBI.

(A) CSF was collected from consecutive, adult nontraumatic control (normal pressure hydrocephalus; NPH) or severe TBI patients. Human ILCs were sorted using forward scatter (FSC)/side scatter (SSC) and identified as CD45⁺, lineage-negative (Lin⁻), CD127⁺ lymphoid cells. Selected populations were analyzed for ILC subtype as follows: ILC1, Lin⁻CD127⁺CD161⁺NKp44⁺; ILC2, Lin⁻CD127⁺GATA3⁺CRTH2⁺; and ILC3, Lin⁻CD127⁺AhR⁺RORγt⁺. ILC functionality was further assessed by cytokine production (ILC1, IFN-γ; ILC2, IL-5/IL-13; and ILC3, IL-17) after cytokine stimulation, as shown. Gray shaded areas indicate isotype controls. (B and C) Quantified data reveal low basal expression of ILC subtypes, with large increases in all ILC classes after TBI. Scatterplots, which are expressed as mean ± SD, depict ILC subtypes as total cell number (B) and % leukocytes (C). Data from individual patients (n = 6 NPH patients, n = 6 severe TBI patients) were compared within each ILC subtype using a 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001 versus sham).

Figure 3. Increased presence of CNS-resident ILCs within the meninges after experimental TBI.

(A) Representative panels depict multiparameter flow cytometry and gating strategy to identify ILCs in meninges at 1 week after sham or TBI in mixed-sex C57Bl/6J mice. ILCs were sorted using forward scatter (FSC)/side scatter (SSC) and identified as CD45⁺, lineage-negative (Lin⁻), CD127⁺ lymphoid cells. ILC subtypes were further defined as the following: ILC1, CD45⁺Lin⁻CD127⁺IL-12Rβ2⁺; ILC2, CD45⁺Lin⁻CD127⁺GATA3⁺; and ILC3, CD45⁺Lin⁻CD127⁺RORγt⁺. Gray shaded areas indicate isotype controls. (B–D) Quantified data showing ILCs subtypes at 1 day (B), 7 days (C), or 1 year (D) after TBI. Quantified data (n = 5 mice/group) are presented as mean ± SD and compared within each ILC subtype using a Student’s t test (*P < 0.05, **P < 0.01, ***P < 0.001 versus sham).

Figure 4. Immunometabolic regulation of ILCs after TBI.

(A) Phosphorylated AMPKα1 (p-AMPKα1), a measure of AMPK activation, was assessed in meningeal cells at 24 hours after sham/TBI in WT mice. Isolated meninges were assessed by forward scatter (FSC)/side scatter (SSC), and selected populations were further analyzed for p-AMPKα. Scatterplots depicting the % total p-AMPKα⁺ cells are indicative of suppressed AMPK activation within the meninges after TBI. (B) AMPKα1–global KO (AMPKα1^–/–) mice showed higher frequencies of all ILC subtypes after TBI, as compared with WT mice, with most pronounced increases noted for ILC1 and ILC3. (C) Intracisternal administration of IL-33 (1 μg) increased meningeal expression of p-AMPKα after TBI, as compared with placebo treatment in both WT mice and in Rag1^–/– mice, which lack mature B and T lymphocytes, but possess functional ILC. Conversely, p-AMPKα was unaffected by IL-33 treatment in Rag1^–/– IL2rg^–/– mice, which lack both mature lymphocytes and ILC2. (D) Intracisternal administration of IL-33 (1 μg) increased meningeal expression of ILC2 and suppressed both ILC1 and ILC3 expansion at day 5 after TBI in WT and Rag1^–/– mice, as compared with placebo (PBS). In contrast, IL-33 did not affect ILC number in Rag1^–/– IL2rg^–/– mice, which lack ILC2. Meningeal tissue was analyzed by flow cytometry. (E) The stimulatory effects of intracisternal IL-33 on ILC2 frequency were lost in AMPKα1^–/– mice, as compared with WT mice. For all panels, quantified data are presented as the mean ± SD from n = 6 mice/group. For each panel, data were compared within each ILC subtype using a 2-tailed Student’s t test (*P < 0.05, **P < 0.01, ****P < 0.0001).

Figure 5. Metformin increased the frequency of ILC2_reg after TBI.

(A) Placebo or Metformin (3 μg) was intracisternally administered at 2 hours after TBI, and isolated meninges were analyzed at day 5 after sham/TBI by forward scatter (FSC)/side scatter (SSC). Lin^–, CD127⁺, GATA3⁺ ILC2s were gated and further analyzed for the expression of the regulatory cytokine IL-10. Representative panels are provided for each group. (B and C) Quantification of ILC subtypes (B), including IL-10⁺ ILC2_reg (C). Data are mean ± SD (n = 4-7 mice/group). Data were compared using a One-Way ANOVA followed by Tukey’s post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).