Transitory presence of myeloid-derived suppressor cells in neonates is critical for control of inflammation

Abstract

Myeloid-derived suppressor cells (MDSCs) are pathologically activated and relatively immature myeloid cells that have been implicated in the immunological regulation of many pathologic conditions. Phenotypically and morphologically, MDSCs are similar to neutrophils (PMN-MDSCs) and monocytes (M-MDSCs). However, they have potent suppressive activity and distinct gene expression profiles and biochemical characteristics. No or very few MDSCs are observed in steady-state physiological conditions. Therefore, until recently, accumulation of MDSCs was considered a consequence of pathological processes or pregnancy. Here, we report that MDSCs with a potent ability to suppress T cells are present during the first weeks of life in mice and humans. MDSC suppressive activity was triggered by lactoferrin and mediated by nitric oxide, PGE2, and S100A9 and S100A8 proteins. MDSCs from newborns had a transcriptome similar to that of tumor MDSCs, but with strong upregulation of an antimicrobial gene network, and had potent antibacterial activity. MDSCs played a critical role in control of experimental necrotizing enterocolitis (NEC) in newborn mice. MDSCs in infants with very low weight, who are prone to NEC, had lower MDSC levels and suppressive activity than did infants with normal weight. Thus, the transitory presence of MDSCs may be critical for regulation of inflammation in newborns.

Figure 1. Expansion of MDSC in newborn mice

a. The proportion and absolute number of the populations of splenocytes at different time after birth (n=8). In AM the total number of splenic cells could not be directly compared with that in NBM due to the much larger spleen size. These results are provided for reference only. b. CD3/CD28 inducible proliferation of CD4⁺ and CD8⁺ T cells in the presence of M-MDSC isolated from newborn (NBM) and adult (AM) mice. No stim - not activated T cells (negative control); No MDSC - T cell proliferation in the absence of MDSC. Proliferation was measure by CFSE dilution. (n=4–6). c. Antigen-specific proliferation of CD8⁺ T cells in the presence of PMN-MDSC isolated from NBM and AM. Proliferation was measure in triplicates by ³H-thymidine uptake. OT-1 T cells were used as responders in antigen-specific suppression assay (cpm =counts per minute). Experiments were performed in triplicates. Individual experiments are shown. Left panel NBM (n=7), right panel AM (n=5). d. Antigen non-specific suppression assays of M-MDSC isolated from NB spleen at different time points after birth (n=4) e. Antigen-specific suppression assays of PMN-MDSC isolated from NB spleen at different time points after birth. (n=4). In all plots mean ± SD are shown. f. s100a8 and s100a9 gene expression in PMN-MDSC from 7-day NBM and AM mice (n=7–14). g. Intracellular S100A9 protein expression measured by flow cytometry in PMN-MDSC or M-MDSC from 7-day NBM or AM mice. Please note the differences in the scale of MFI between PMN-MDSC and M-MDSC (n=6–11). h. Antigen specific suppression assays of PMN-MDSC and M-MDSC isolated from spleens of 7-day old S100A9 KO mice. Cell proliferation was measured in triplicates by ³[H]-thymidine uptake. Typical example of three experiments is shown. i. Ptges gene expression in PMN-MDSC (qRT-PCR). NBM (n=7), NB S100A9 KO (n=7), AM control (n=14). j. Amount of PGE2 measured by ELISA in cell supernatants from 24hr culture of PMN-MDSC from adult and NB (3 or 7 days old) WT mice or S100a9 KO 7 days old NB mice (n=4). k. Antigen specific suppression assay of PMN-MDSC isolated from spleens of 7 day-old NB WT and Cox2 KO mice (n=4). In all plots mean ± SD are shown. In most panels p values are shown on the graphs.

Figure 2. Lactoferrin is responsible of accumulation of MDSC in newborn mice

a. Proportion of monocytes and neutrophils in LF treated 3 weeks old mice (n=8). Control – mice treated with PBS. b. Suppression of CD3/CD28 stimulated T cells by M-MDSC isolated from AM treated with LF or PBS (control) (n=4) c. Antigen-specific suppression assay of PMN-MDSC isolated from AM treated with LF (n=6). d. Expression of nos2 in MDSC from LF treated mice (n=6) (left panel). Amount of NO in M-MDSC from LF treated mice (n=6) (right panel). e. Suppression of CD3/CD28 stimulated T cells by M-MDSC from LF-treated mice in the presence of inhibitors of ROS (NAC), arginase-I (nor-NOHA), or Nos2 (L-NMMA) (n=6). f. Expression of s100a9 and s100a8 genes in PMN-MDSC from LF treated mice (n=3). g. Mean fluorescence intensity (MFI) of intracellular expression of S100A9 protein detected by flow cytometry in PMN-MDSC from AM treated with LF (n=6); Inset – amount of S100A9 protein detected by Western Blot. Representative result of three independent experiments is shown. h. PGE2 amount in cell lysates from 24hr culture of PMN-MDSC isolated from AM treated with LF (n=6). i. Antigen-specific T cell suppressive activity of PMN-MDSC isolated from LF treated wild-type and S100A9 KO mice. Proliferation was measure by ³[H]-thymidine uptake in triplicates. Typical example of three performed experiments is shown. Dotted line – the level of T cell proliferation in the absence of MDSC. j. Antigen specific suppression assays of PMN-MDSC isolated from LF KO mice. Experiment was performed in triplicates. Results of 5 experimtnts are shown. k. Mean fluorescence intensity (MFI) of intracellular S100A9 protein expression in PMN-MDSC in AM (n=11),NB WT (n=10), and LFKO (n=8) mice. l. Expression of ptges in PMN-MDSC from AM (n=13), NB WT (n=7), and LF-KO (n=5) mice. m. Amount of PGE2 measured by ELISA in cell lysates from 24hr culture of PMN-MDSC from AM (n=4) and NBM WT mice (n=2) or LF KO NBM (n=2). In all plots mean ±SEM are shown. P values in two-tailed Student’s test are shown on the graphs.

Figure 3. MDSC in human infants

a. Proportion of MDSC in peripheral blood of infants. PMN-MDSC (left panel) and M-MDSC (right panels) were evaluated in PBMC of peripheral blood of infants with normal (NW) and very low weight (VLW) at indicated time after birth (n=6). Individual data and SD are shown. b. Functional activity of MDSC from infants. M-MDSC and PMN-MDSC were sorted from PBMC of infants with normal weight (NW) and very low weight (VLW) and added to T cells isolated from blood and stimulated with CD3/CD28 antibody. Proliferation was measured in triplicates using CFSE labeling (n=3). c–e. LOX-1⁺ PMN-MDSC in newborns. c. LOX-1⁺ and LOX-1⁻ CD15⁺ cells were isolated from peripheral blood of newborn infants and tested in suppressive activity against CD3/CD28 stimulated T cells. Proliferation was measured in triplicates using CFSE staining (n=3). d. Proportion of LOX-1⁺ cells among CD15⁺ neutrophils in infants of different age, 1–2 days (n=10), 3–7 days (n=18), adults (n=7). Individual results and SD are shown. e. Proportion of LOX-1⁺ cells in NW (n=8) and VLW (n=4) infants of different age. Individual results are shown. f–l. Mechanisms regulating MDSC activity in infants. Samples of blood were collected from newborn infants 1–5 days after birth. M-MDSC and PMN-MDSC were sorted from PBMC. f. Suppressive activity of M-MDSC from NW infants in the presence of inhibitors of ROS (NAC), arginase I (nor-NOHA) or NOS2 (L-NMMA). Effectors were CD4⁺ or CD8⁺ T cells stimulated with CD3/CD28 antibodies. Proliferation was measured in triplicates using CFSE staining (n=3). g. Expression of NOS2 in M-MDSC (n=3). h. Nitrites produced by M-MDSC. NW (n=6), VLW (n=3), control cells from adults (n=6). Individual results, meant and SD are shown. i. The amount of PGE2 produced by PMN-MDSC and measured by ELISA in cells lysates. NW and adults - n=8, VLW - n=3. Individual results, mean and SD are shown. j. Expression of S100A8 and S100A9 in PMN-MDSC and M-MDSC measured by qPCR (n=3). k. The amount of S100A9 protein in cell lysates of PMN-MDSC. NW - n=8, VLW - n=3 adults - n=8. l. Expression of LTF in PMN-MDSC and M-MDSC (n=3). m. LOX-1⁺ PMN-MDSC in blood of 5 days old infants received formula (n=11) or mother’s milk (n=6). In all panels, mean and SD are shown. P values in two-tailed Student’s test are shown on the graphs.

Figure 4. MDSC role in Necrotizing Enterocolitis (NEC) experimental model

a. Representative images of mice intestine after NEC induction. Numbers in the pictures represent NEC severity from 0 (normal epithelium) to IV (most severe NEC). b. Detection of FD7000 (directly proportional to intestine permeability) (n=13), c. small intestine inflammation score (n=15), d. survival (n=24) in one day and four days old mice after NEC induction. e. Bacterial load in small intestine of mice evaluated using 16s rRNA gene abundance analysis (n=8); f. Flow cytometry analysis of MDSC presence in lamina propria (LP) after MDSC depletion with DR5 antibody (n=6). g. Survival analysis of WT NEC mice with MDSC depletion by DR5ab (n=13) or treated with IgG (n=10). h. Small intestine inflammation score in 4 days old mice after MDSC depletion (n=6). i. FD7000 after MDSC depletion (n=6). j,k. Bacterial load was evaluated in intestine (n=6) (j) and blood (n=6) (k). Bacterial load was normalized to the level in NBM without NEC. Values for individual mice are shown. l. Inflammation score in small intestine of NBM after treatment with exogenous Gr1⁺ cells from NBM, adult mice (AM) or vehicle control (PBS) (n=15). m. intestine permeability (n=15); n. Survival of mice after NEC induction and treatment with Gr1⁺ cells from NBM (n=20), AM (n=15) or vehicle control (PBS) (n=15). o. Bacterial load was evaluated in intestine and blood (n=6). p. Flow cytometry analysis of MDSC presence in lamina propria (LP) of Rag1KO mice with induced NEC treated with DR5 antibody or control IgG (n=6). r. Small intestine inflammation score of Rag1KO mice with induced NEC treated with DR5 antibody (n=6) s. FD7000 in mice treated with DR5 antibody (n=6). t. Survival analysis of mice treated with DR5 antibody or IgG after NEC induction (n=11). Individual values and SD are presented. Results of individual mice are shown. In all panels, mean and SD are shown. P values in two-tailed Student’s test are shown on the graphs.