IL-11 induces NLRP3 inflammasome activation in monocytes and inflammatory cell migration to the central nervous system

Abstract

The objective of this study is to examine IL-11-induced mechanisms of inflammatory cell migration to the central nervous system (CNS). We report that IL-11 is produced at highest frequency by myeloid cells among the peripheral blood mononuclear cell (PBMC) subsets. Patients with relapsing-remitting multiple sclerosis (RRMS) have an increased frequency of IL-11⁺ monocytes, IL-11⁺ and IL-11R⁺ CD4⁺ lymphocytes, and IL-11R⁺ neutrophils in comparison to matched healthy controls. IL-11⁺ and granulocyte-macrophage colony-stimulating factor (GM-CSF)⁺ monocytes, CD4⁺ lymphocytes, and neutrophils accumulate in the cerebrospinal fluid (CSF). The effect of IL-11 in-vitro stimulation, examined using single-cell RNA sequencing, revealed the highest number of differentially expressed genes in classical monocytes, including up-regulated NFKB1, NLRP3, and IL1B. All CD4⁺ cell subsets had increased expression of S100A8/9 alarmin genes involved in NLRP3 inflammasome activation. In IL-11R⁺-sorted cells from the CSF, classical and intermediate monocytes significantly up-regulated the expression of multiple NLRP3 inflammasome-related genes, including complement, IL18, and migratory genes (VEGFA/B) in comparison to blood-derived cells. Therapeutic targeting of this pathway with αIL-11 mAb in mice with RR experimental autoimmune encephalomyelitis (EAE) decreased clinical scores, CNS inflammatory infiltrates, and demyelination. αIL-11 mAb treatment decreased the numbers of NFκBp65⁺, NLRP3⁺, and IL-1β⁺ monocytes in the CNS of mice with EAE. The results suggest that IL-11/IL-11R signaling in monocytes represents a therapeutic target in RRMS.

Keywords: EAE; IL-11; NLRP3 inflammasome; monocyte; multiple sclerosis.

Fig. 1.

IL-11 and IL-11RA have the highest expression in myeloid cells. (A) The percentages of IL-11⁺ and IL-11R⁺ cells in 7 gated PBMC cell subsets from 14 RRMS patients. (B) The percentages of IL-11⁺ and IL-11R⁺ cells from 9 RRMS patients in comparison to matched HCs. (C) Phenotype of IL-11⁺ CD14⁺ monocytes, CD4⁺ cells, and neutrophils. (D) IL-11R⁺ CD14⁺ monocytes and CD4⁺ lymphocytes in 9 RRMS patients.

Fig. 2.

IL-11⁺ and GM-CSF⁺ monocytes, CD4⁺ cells, and neutrophils accumulate in the CSF of RRMS patients. (A) The frequency of cytokine-expressing CD14⁺ cells and IL-11⁺CD14⁺ monocytes coexpressing inflammatory markers; (B) CD4⁺ cells; and (C) neutrophils was determined in paired PBMC and CSF samples from 9 RRMS patients.

Fig. 3.

IL-11 induces the expression of NLPR3 inflammasome genes in monocytes. (A) Schematic representation of the experiment. Blood samples were sorted for IL-11R⁺ cells, incubated with/without IL-11 before scRNA sequencing (n = 3 RRMS patients). (B) The Uniform Manifold Approximation and Projection (UMAP) clustering in 29 immune cell types. (C) A cluster-defining gene expression in monocytes and CD4⁺ T cell subsets. (D) DEGs following IL-11 stimulation in classical monocytes, volcano plot. The expression level of some biologically relevant significantly up-regulated genes in classical monocytes, violin plots. Gene ontology and pathway analysis for IL-11-up-regulated genes in monocytes. (E) DEGs following IL-11 stimulation in Th1/Th17 CD4⁺ cells, volcano plot; up-regulated genes in Th1/Th17 cells in violin plots. Gene ontology and pathway analysis results for up-regulated genes in all CD4⁺ cell types.

Fig. 4.

CSF-derived IL-11R⁺ monocytes up-regulate genes involved in cell migration. (A) The UMAP cell clustering in 29 immune cell types. (B) A cluster-defining gene expression in monocyte subsets. (C) DEGs between CSF and blood samples in classical monocytes, volcano plot; up-regulated genes in CSF in classical monocytes, violin plots. (D) DEGs in intermediate monocytes, volcano plot; the expression level of selected up-regulated genes in CSF intermediate monocytes, violin plots. (E) Gene ontology and pathway analysis for up-regulated genes in all monocyte subsets.

Fig. 5.

IL-11 induces NLPR3 inflammasome–related proteins in RRMS. (A) Magnetic bead–separated monocytes from RRMS patients (n = 11) were treated with rhIL-11, and the expression of indicated genes was measured by qRT-PCR. (B and C) PBMCs from the same patients were treated with rhIL-11, and the expression of S100A8/9 was measured in gated CD3⁺CD4⁺ T cells and intracellular cytokines in monocyte subsets.

Fig. 6.

αIL-11 mAb suppresses RREAE via inhibition of NLRP3 inflammasome activation. (A) A time course flow cytometry study during the RREAE (6 mice per group). (B) The frequencies of IL-11⁺ and IL-11R⁺ cells in PBMC and CNS-infiltrating cells from the same mice, 21 d.p.i. (C) Twelve SJL mice were immunized with PLP_139-151 to induce RREAE and were treated with αIL-11 mAb or IgG2A isotype control i.p starting at the onset of disease for 10 consecutive days. The presented data are representative of three independent experiments with at least 6 mice per group. (D) IL-11 was measured in plasma on day 21 p.i. (n = 14 per group). (E) On 18 d.p.i., H&E and myelin basic protein (MBP) staining quantified inflammatory foci and demyelinated areas. (F) Number of IL-11⁺Iba-1⁺ cells and IL-11⁺CD4⁺ cells (arrows) averaged from at least 8 microscope fields within the lesion (n = 5 mice per group, scale bar, 100 µm; 10 µm for higher magnification). (G–K) At day 21 p.i., RREAE mice treated with αIL-11mAb or isotype control were killed, and cell suspension from PBMCs, LN, spleen, and CNS infiltrates was used for flow cytometry (n = 6 or 12 per group). (G) Intracellular cytokine staining in PBMCs and CNS (n = 6). (H) CD4⁺ cells from CNS infiltrates. (I) The frequency of IL-11⁺CD4⁺ (PBMC) and IL-11R⁺CD4⁺ (LN) cells (n = 6 for IL-11 and n = 12 for IL-11R). (J) The percentages of CCR6⁺CD4⁺ cells in PBMCs, LNs, spleen, and CNS, and (K) CXCR3⁺CD4⁺ cells in PBMCs. (L) IL-11R⁺ monocytes were isolated from the spleen of RREAE mice and treated with rmIL-11. Inflammasome-related gene expression was determined by RT-PCR (n = 4). (M) IL-11R⁺ monocytes from the spleen of isotype-control or αIL-11 mAb–treated mice with RREAE, the expression of NLRP3-related genes (n = 7), and the number of monocytes expressing inflammasome-related proteins in the CNS infiltrates (n = 7).