Lactoferrin-induced myeloid-derived suppressor cell therapy attenuates pathologic inflammatory conditions in newborn mice

Abstract

Inflammation plays a critical role in the development of severe neonatal morbidities. Myeloid-derived suppressor cells (MDSCs) were recently implicated in the regulation of immune responses in newborns. Here, we report that the presence of MDSCs and their functional activity in infants are closely associated with the maturity of newborns and the presence of lactoferrin (LF) in serum. Low amounts of MDSCs at birth predicted the development of severe pathology in preterm infants - necrotizing enterocolitis (NEC). In vitro treatment of newborn neutrophils and monocytes with LF converted these cells to MDSCs via the LRP2 receptor and activation of the NF-κB transcription factor. Decrease in the expression of LRP2 was responsible for the loss of sensitivity of adult myeloid cells to LF. LF-induced MDSCs (LF-MDSCs) were effective in the treatment of newborn mice with NEC, acting by blocking inflammation, resulting in increased survival. LF-MDSCs were more effective than treatment with LF protein alone. In addition to affecting NEC, LF-MDSCs demonstrated potent ability to control ovalbumin-induced (OVA-induced) lung inflammation, dextran sulfate sodium-induced (DSS-induced) colitis, and concanavalin A-induced (ConA-induced) hepatitis. These results suggest that cell therapy with LF-MDSCs may provide potent therapeutic benefits in infants with various pathological conditions associated with dysregulated inflammation.

Keywords: Cellular immune response; Immunology; Inflammation; Monocytes; Neutrophils.

Figure 1. Clinical significance of MDSCs in infants.

(A) Percentages of Lox1⁺CD15⁺ PMN-MDSCs in adults (AD) (n = 10), full-term (term) (n = 8), and preterm (n = 11) infants and patients with NEC (n = 14). Samples from NEC patients were collected at diagnosis (6 to 66 days after birth). In all other cases, samples were collected between days 1 and 4 after birth. (B) Percentages of PMN-MDSCs in infants who later developed or did not develop NEC. Samples were collected on days 1–3 (AD, n = 10; term, n = 8; preterm, no NEC, n = 51; preterm with NEC, n = 9) or 4–7 (term, n = 9; preterm, no NEC, n = 15; preterm with NEC, n = 5) after birth. (C) Correlation between proportion of PMN-MDSCs and body weight of infants at different age. (D) Functional activity of PMN-MDSCs from infants using T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate with CFSE labeling (n = 4–8). (E) Expression of indicated genes in PMN-MDSCs measured by quantitative reverse-transcriptase PCR (qRT-PCR) (n = 4). (F) Antibacterial activity of PMN-MDSCs against E. coli (n = 4). (G) Concentrations of LF in plasma from adults and newborns determined by ELISA (n = 8–11). (H) Correlation between LF concentration (plasma) and proportion of PMN-MDSCs, body weight, or gestational age of newborns. (I) LF concentration in plasma of infants who were fed with breast milk (n = 13) or formula (n = 4). In all panels, individual results and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests (E, F, and I) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test (A, B, D, and G). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. For C and H, Spearman’s correlation coefficient was calculated, and actual P values are shown.

Figure 2. LF converts PMN and MON to MDSCs.

(A) BM cells from 2-week-old mice were treated with LF or vehicle (PBS) in the presence of GM-CSF for 2 days. Typical example of flow cytometry (left), absolute numbers, and proportion of PMN and MON (right) (n = 6). (B) Suppressive activity of LF-induced PMN-MDSCs against proliferation of OT-I–derived CD8⁺ T cells stimulated with cognate peptide (n = 6). Nonstimulated T cells (No-stim) and stimulated T cells without PMN (No-PMN) were used as controls. (C) Absolute numbers of CD11b⁺HLA-DR^–/lo cells in PBS- or LF-treated CB mononuclear cells for 2 days (n = 6). (D) Suppression of LF-induced CD11b⁺HLA-DR^–/lo cells from CB mononuclear cells against T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate by CFSE dilution (n = 6). (E) PGE2 and NO in myeloid cells from PBS- or LF-treated CB mononuclear cells (n = 6). (F) Absolute numbers of myeloid cells in PBS- or LF-treated preterm infant–derived CB mononuclear cells for 2 days (n = 6). (G) CD11b⁺HLA-DR^–/lo were sorted from PBS- or LF-treated preterm infant CB mononuclear cells, followed by incubation with T cells stimulated with CD3/CD28 antibodies. T cell proliferation was measured in triplicate by CFSE labeling (n = 6). In all plots, the results of individual experiments and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests (A and C–G) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test (B). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3. Antibacterial activity of LF-MDSCs.

(A and B) Antibacterial activity of PMN-MDSCs or M-MDSCs isolated from PBS- or LF-treated newborn BM cells against E. coli (A, n = 6) or with C. albicans (B, n = 3–6). CFU were calculated after 24 hours of incubation. Medium (Med) alone was used as control. (C) Antibacterial activity of PMN-MDSCs and M-MDSCs against C. sakazakii (n = 6). (D) Phagocytosis of FITC-labeled pHrodo E. coli BioParticles (100 μg/1 × 10⁵ cells for 90 minutes) by LF-induced mouse PMN-MDSCs or M-MDSCs. Typical example (left) and cumulative results (right) are shown (n = 6). (E) Phagocytosis of FITC-labeled pHrodo E. coli BioParticles by LF-induced human MDSCs from CB (n = 6). (F) Antibacterial activity of myeloid cells from PBS- or LF-treated CB mononuclear cells (n = 3–6). Medium alone was used as control. In all plots, the results of individual experiments and mean ± SD are shown. P values were calculated using 2-sided Student’s t tests (D and E) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test (A–C and F). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4. LF regulates MDSC function via NF-κB transcription factor.

(A) Western blot of p65 in cytoplasm (Cyt) and nuclei (Nuc) in MDSCs from newborn mouse BM cells treated with LF and/or NF-κB inhibitor (JSH23, 2.5 μM). Control cells were treated with PBS. Typical example of 3 performed experiments is shown. (B) Typical example of staining (on the left) and absolute numbers of PMN-MDSCs and M-MDSCs (right) from mouse BM cells treated with LF and/or JSH23 (n = 6). (C) Suppressive activity of PMN-MDSCs (n = 6). T cell proliferation of OT-I CD8⁺ T cells stimulated with SIINFEKL was measured in triplicate by CFSE staining. Mean ± SD are shown. (D) The amount of S100A9 in PMN-MDSCs measured by ELISA. (E) The amount of PGE2 in PMN-MDSCs measured by ELISA (n = 6). (F) Absolute numbers of myeloid cells from LF-treated CB mononuclear cells in the presence or absence of JSH23 (n = 6). (G) Suppressive activity of myeloid cells from LF-treated CB cells in the presence or absence of JSH23. Effectors were CD4⁺ or CD8⁺ T cells stimulated with CD3/CD28 antibodies. Proliferation was measured in triplicate by CFSE staining. Results of individual experiments are shown (n = 4). In all plots, data represent mean ± SD. P values were calculated using 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 5. The effect of LF on myeloid cells is mediated by the LRP2 receptor.

(A) Expression of indicated LF receptors in MDSCs from 2-week-old mice measured by qRT-PCR (n = 6). (B) Expression of Lrp2 in myeloid cells from mice of different ages measured by qRT-PCR (n = 6). (C) Surface expression of Lrp2 on mouse myeloid cells measured by flow cytometry (n = 6). (D) Expression of LF receptors in myeloid cells from PB (PBMC) or CB in full-term infants measured by qRT-PCR (n = 5). (E) Expression of ITLN1 and LRP2 in myeloid cells from PB of adults (AD) and full-term, preterm, and NEC infants or CB measured by qRT-PCR (n = 5–6). (F–K) Silencing of Itln1 and Lrp2 in newborn mouse BM cells with shRNA or scramble shRNA (control) lentiviruses, followed by treatment with PBS or LF for 2 days. (F) The absolute numbers of PMN-MDSCs and M-MDSCs measured by flow cytometry (n = 4–6). (G) Suppressive activity of PMN-MDSCs. Effectors were OT-I CD8⁺ T cells stimulated with SIINFEKL. T cell proliferation was measured in triplicate by CFSE staining (n = 3). Upper panel shows typical example. Lower panel shows cumulative results. (H) Suppressive activity of M-MDSCs. Effectors were CD4⁺ or CD8⁺ T cells stimulated with CD3/CD28 antibodies (n = 3). Left panel shows typical example. Right panel shows cumulative results. (I) The amount of S100A9 in PMN-MDSCs measured by ELISA. (J) The amount of PGE2 in PMN-MDSCs measured by ELISA. (K) The amounts of nitrites in M-MDSCs measured by the Nitrite Assay Kit. (I–K, n = 6) Data represent individual results and mean ± SD. P values were calculated using 2-sided Student’ s t tests (F–K) or 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test (A–E). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 6. Therapeutic effect of LF-MDSCs in the model of NEC.

(A–E) NEC was induced in 1- to 3-day-old mice as described in Supplemental Figure 9A. (A) Proportion of CFSE-labeled donor CD11b⁺Gr1⁺ cells in small intestine lamina propria (SILP) of recipients (n = 6). (B) Representative H&E staining of mouse intestine after NEC induction with or without transfer of MDSCs (left). Inflammation scores were calculated based on the severity of NEC (right) (n = 8). (C) FITC-labeled dextran (FD70) (reflecting intestine permeability) after NEC induction with or without transfer of MDSCs (n = 6). (D) Survival of mice after NEC induction with or without transfer of MDSCs (n = 28 in each group). (E) Bacterial load in small intestine and blood after NEC induction with or without transfer of cells from PBS- or LF-treated BM cells (n = 6). (F–I) NEC was induced in 7-day-old mice treated with LF protein or transfer with LF-MDSCs. (F) Inflammation scores in the small intestine after NEC was induced and treated with LF protein or transfer of LF-MDSCs. (G) Detection of FITC-labeled dextran (FD70) after NEC was induced and treated with LF protein or transfer of LF-MDSCs (n = 6). (H) Survival of mice after NEC was induced and treated with LF protein or transfer of LF-MDSCs. Control, n = 40; NEC, n = 37; LF, n = 40; LF-MDSCs, n = 40. (I) Bacterial load in small intestine (left) and blood (right) after NEC was induced and treated with LF protein or transfer or LF-MDSCs (n = 6). In all plots, data represent mean ± SD. P values were calculated using 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test (A–C, E–G, and I). *P < 0.05; **P < 0.01; ***P < 0.001. Survival statistics were calculated using log-rank (Mantel-Cox) test.

Figure 7. Therapeutic effect of LF-MDSCs under inflammatory conditions.

(A and B) Lung inflammation induced by OVA in 6- to 8-week-old adult mice. (A) Representative H&E images of lungs (left) and inflammation scores (right) after lung inflammation induction with or without transfer of LF-MDSCs (n = 6). (B) Percentages of eosinophils in BALF (n = 9). (C–E) DSS-induced model of colitis in 6- to 8-week-old adult mice. (C) H&E staining of colon (left) and histology scores (right) after DSS-induced colitis, with or without transfer of LF-MDSCs. n = 7. (D and E) DAI (D) and loss of body weight (E) after colitis induction with or without transfer of exogenous LF-MDSCs were measured (n = 6). (F and G) Model of ConA-induced hepatitis in 6- to 8-week-old adult mice. (F) H&E staining of liver after induction of hepatitis with or without transfer of exogenous LF-MDSCs. (G) Levels of ALT (left) and AST (right) in serum (n = 6). In all plots, data represent individual results and mean ± SD. P values were calculated using 1-way ANOVA followed by Tukey-Kramer multiple-comparisons test (A, B, C, and G). ***P < 0.001.