Autonomous TNF is critical for in vivo monocyte survival in steady state and inflammation

Abstract

Monocytes are circulating mononuclear phagocytes, poised to extravasate to sites of inflammation and differentiate into monocyte-derived macrophages and dendritic cells. Tumor necrosis factor (TNF) and its receptors are up-regulated during monopoiesis and expressed by circulating monocytes, as well as effector monocytes infiltrating certain sites of inflammation, such as the spinal cord, during experimental autoimmune encephalomyelitis (EAE). In this study, using competitive in vitro and in vivo assays, we show that monocytes deficient for TNF or TNF receptors are outcompeted by their wild-type counterpart. Moreover, monocyte-autonomous TNF is critical for the function of these cells, as TNF ablation in monocytes/macrophages, but not in microglia, delayed the onset of EAE in challenged animals and was associated with reduced acute spinal cord infiltration of Ly6C^hi effector monocytes. Collectively, our data reveal a previously unappreciated critical cell-autonomous role of TNF on monocytes for their survival, maintenance, and function.

Figure 1.

Absence of TNF signaling impairs steady-state monocytes. (A) Scheme illustrating monocyte development (top) and heat map of RNA sequencing expression data of NF-κB–driven genes in sorted MDP, cMOP, and Ly6C^hi BM monocytes, as well as Ly6C^hi, Ly6C^int, and Ly6C^lo blood monocytes (bottom). (B) RNA sequencing reads for tnf, tnfrsf1a, and tnfrsf1b expression throughout sequential monocyte development stages in A. n = 2–3. (C) FACS analysis of T cells and Ly6C^hi and Ly6C^lo monocytes (MO) extracted from blood of either [TNFR1^−/−/WT > WT] (top) or [TNFR1/2^−/−/WT > WT] (bottom) mixed BM chimeras. (D) Quantification of myeloid cells and precursors. n = 3–5. Mann-Whitney U test was used. (E) FACS analysis of Ly6C^hi and Ly6C^lo monocytes extracted from blood of [TNF/WT > WT] mixed BM chimeras. (F) Quantification of myeloid cells and precursors in C and D. n = 5–10. Student’s t test was used. *, P < 0.05.

Figure 2.

Absence of TNF signaling increases apoptosis of circulating monocytes. (A) Scheme of in vitro co-culturing of TNF/TNFR1-deficient and WT monocytes in microwells. (B) Traces of co-cultured WT (TNFR1^+/+) and TNFR1^−/− monocytes monitored in culture for 48 h. WT/TNFR1^−/− cells were distinguished by fluorescently labeled antibodies against CD45.1/CD45.2, respectively. Cell death time was monitored using PI. Bar, 10 μm. (C and D) Cumulative fraction of dead monocytes (PI⁺) during 48 h (C) and timing of PI signal (indicating time of cell death) for individual cells (D) in co-cultures of: Ly6C^hi WT/TNFR1^−/− monocytes (n = 567 and 960 cells, respectively), Ly6C^hi WT/TNF^−/− (n = 641 and 1,348), Ly6C^lo WT/TNFR1^−/− (n = 1,567 and 1,766), and Ly6C^lo WT/TNF^−/− (n = 1,121 and 1,162). Red lines in D show means. **, P < 0.01; ***, P < 0.001. Mann-Whitney U test was used. (E) Fraction of monocytes surviving after 48 h of co-culture. ***, P < 0.001. χ² test was used. (F) Cumulative fraction of dead (PI⁺) Ly6C^hi WT/TNF^−/− monocytes co-cultured in microwell arrays during 48 h without TNF (n = 80 and 164) or with 2.5 ng/ml (n = 192 and 142) or 50 ng/ml (n = 112 and 181) recombinant TNF (rTNF). Closed and open circles show data (WT and TNF^−/−, respectively), and continuous and dashed lines represent the best-fit logistic curve (see Materials and methods). The table shows t_half, the time by which the curves reach half of their maximum value. (G) FACS analysis of mixed cultures of MACS-sorted CD115⁺CD11b⁺ BM cells of CD45.1 and CD45.1/CD45.2 TNFR1^−/− mice in the presence of 10 ng/ml M-CSF. Data are representative of three biological repeats. rM-CSF, recombinant M-CSF. (H and I) Timing of PI signal for individual cells (H) and fraction of cells surviving after 48 h (I) of cMOPs sorted from BM of WT and TNF^−/− mice, co-cultured in microwell arrays. n = 47 and 170. (J and K) Fraction of annexin V⁺ Ly6C^lo monocytes in BM chimeric [TNF/WT > WT] (F) and [TNFR1^−/−/WT > WT] (G). n = 9–10. ***, P < 0.001. Paired Student’s t test was used.

Figure 3.

Total monocyte/macrophage TNF deletion affects EAE onset. (A) Gating strategy for spinal cord–infiltrating effector monocytes, used for all cell sorting and analysis. (B) Volcano plot performed for comparison of sorted steady-state blood monocytes and effector monocyte infiltrates in the spinal cord at EAE day 9. Numbers indicate genes significantly up-regulated (red) or down-regulated (green) in effector monocytes compared with steady-state monocytes (log difference >1 or <−1; P < 0.05). (C) Canonical pathway analysis performed for genes significantly changed in D. Z-score >2 or <−2. iNOS, inducible nitric oxide synthase; NO, nitric oxide; PLK, polo-like kinase; PPAR, peroxisome proliferator-activated receptor; ROS, reactive oxygen species. (D) Normalized reads of tnf, tnfsr1a, tnfrsf1b, and emr1 expression in steady state and effector monocytes, as in Fig. 2 E. n = 3–4. Mann-Whitney U test was used. (E) ELISA of sera collected from tnf^fl/fl and cx₃cr1^Cre:tnf^fl/fl mice 4 h after intraperitoneal challenge with 200 µg LPS in vivo. n = 5. Mann-Whitney U test was used. (F) TNF production by myeloid cells in spinal cords of tnf^fl/fl and cx₃cr1^Cre:tnf^fl/fl mice at day 24 after MOG_35–55 immunization, analyzed by FACS. n = 7–4. Mann-Whitney U test was used. (G–I) EAE disease course (G), day of onset (H), and individual maximal score (I) in tnf^fl/fl and cx₃cr1^Cre:tnf^fl/fl mice immunized with MOG_35–55 peptide. n = 7–8. Student’s t test was used. Data are representative of three independent experiments. *, P < 0.05; **, P < 0.01. Results are expressed as means ± SEM.

Figure 4.

Effector monocytes are reduced because of absence of autonomous TNF. (A) FACS analysis of effector monocytes gated as in Fig. 3 A from MOG_35–55 immunized in tnf^fl/fl and cx₃cr1^Cre:tnf^fl/fl mice at day 24. n = 4–7. Mann-Whitney U test was used. (B and C) FACS analysis of spinal cord CD45^hiLy6C^hi infiltrate in MOG_35–55 immunized in tnf^fl/fl and cx₃cr1^Cre:tnf^fl/fl mice at day 9 (n = 5–7; B) or 24 (n = 4–7; C) after immunization. Mann-Whitney U test was used. (D) FACS analysis circulating steady-state (s.s.) Ly6C^hi monocytes (gated as in Fig. S1 B) compared with spinal cord effector monocytes (gated as in Fig. 3 A) at day 9 after MOG_35–55 immunization in [CD45.1 cx₃cr1^gfp:tnf^+/+/CD45.2 cx₃cr1^Cre:tnf^fl/fl > CD45.2 WT] chimeric mice. Resident CD45.2⁺ microglia (gated as in Fig. S3 B) are presented as reference cells for CD45.2 signal. n = 6. Student’s t test was used. *, P < 0.05; ***, P < 0.001.