Subcutaneous delivery of mesenchymal stromal cells induces immunoregulatory effects in the lymph node prior to their apoptosis

Abstract

Background: Mesenchymal stromal cell (MSC) therapy commonly involves systemic infusion of MSCs, which undergo apoptosis in the lung and induce immunoregulatory macrophages that reduce disease. The relevance of this mode of action, however, is yet to be determined for MSCs administered via other routes. Here, we administered MSCs via subcutaneous (SC) injection into inflamed tissue and investigated the immunomodulatory effects on the local lymph node (LN), which is a major site for the initiation and regulation of immune responses.

Methods: A mouse model of localised skin inflammation was established with low-dose lipopolysaccharide (LPS) to in vivo prime adipose-derived MSCs delivered via SC injection. We then analysed the immunomodulatory changes in the LN draining the inflamed tissue, as well as the neutrophil TNF response to LPS re-exposure.

Results: When administered directly into the inflamed skin tissue, SC MSC injection induced an expansion of IL-10-producing MerTK⁺ subcapsular sinus macrophages and T cell zone macrophages, as well as activated CD44⁺ regulatory T cells (Tregs), in the draining LN, which was not observed in the non-draining LN. SC injection of viable, but not apoptotic, MSCs dampened TNF production by inflammatory cells in the draining LN when re-exposed to the inflammatory stimulus. SC injection of MSCs remote to the site of inflammation also did not attenuate the LN response to subsequent inflammatory challenge.

Conclusions: MSCs delivered directly into inflamed skin activated immunoregulatory cells in the local LN and inhibited LN responsiveness to subsequent inflammatory challenge. The immunoregulatory effects of SC-injected MSCs in the LN require priming by inflammatory cytokines in the local milieu. Furthermore, SC-injected MSCs exert anti-inflammatory effects in the draining LN prior to their apoptosis, in contrast to intravenously delivered MSCs, where anti-inflammatory effects are linked to their apoptosis.

Keywords: Inflammation; Lymph nodes; Macrophages; Mesenchymal stem cells.

Fig. 1

SC injected MSCs display a longer dwell time at the site of injection. (A), MSCs were transduced with a firefly luciferase (fluc) vector that also encodes eGFP (fluc-GFP) to enable the quantification of transduction efficiency (indicated by %flu-GFP⁺) by flow cytometry. The cell suspension prepared for injection was adjusted to ensure 1 × 10⁶ fluc-GFP⁺ MSCs per injection. (B) Representative bioluminescent images of luciferase-expressing MSCs after IV, IP and SC injections, with bar graphs present the changes in the radiance (n = 3 mice per group). (C) In vivo bioluminescent signal titration: various number of luciferase-expressing MSCs were injected via SC route into the hock to examine the lower threshold of the detection range. Data expressed as mean ± SEM; n = 3 mice per group

Fig. 2

Establishing an acute inflammation model with low-dose LPS. (A) C57BL/6 mice were SC-injected with 30 ng or 100 ng LPS in 50 µl PBS in the left hock. At 0, 4, 8, 24 h post-LPS injection, skin tissue around the LPS injection site was excised and homogenised for cytokine analysis. Changes in pro-inflammatory TNF, IL-1β and MCP-1 in the skin tissues over the time course. n = 3 mice per timepoint from one experiment. (B) Draining LNs were harvested from mice at 0, 4, 8, 24 h after SC injection of 30 ng LPS in 50 µl PBS in the left hock. Total LN, neutrophil and monocyte cellularity at various timepoints post-LPS injection. (C) Inflammatory gene expression in whole LN cell lysate and sorted LN neutrophils and monocytes at 24 h post-LPS injection. Fold-change differences in mRNA expression of inflammatory genes were plotted after normalising mRNA expression level to housekeeping genes (GAPDH and β-actin). Data expressed as mean ± SEM; UNT (untreated LN) n = 2–3 mice per timepoint; LPS (LPS dLN) n = 3–4 mice per timepoint

Fig. 3

SC MSC injection expands IL-10-producing MerTK⁺ macrophages in the LN. (A) C57BL/6 mice were SC-injected with 30 ng LPS in 50 µl PBS in the left hock, and 4 h later 1 × 10⁶ human adipose MSCs in the same hock. Draining (dLN; white bar) and non-draining LNs (ndLN; shaded bar) were harvested 20 h after MSC injection for analysis. (B) Representative flow cytometry plots showing the gating strategies for delineating myeloid and lymphoid populations in the mouse LNs. (C) Changes in total cellularity and cell number of different immune populations in LNs following SC MSC injection. Data expressed as mean ± SEM; n = 4–5 mice per group, representative of 2 independent experiments. One-way ANOVA, Tukey’s multiple comparison, comparing UNT, LPS and MSC dLNs; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (D) Changes in cell number of different LN-resident macrophage subpopulations following MSC treatment. (E) Representative flow cytometry plots showing the identification of IL-10⁺ populations in LN macrophage subpopulations. (F) Changes in IL-10⁺ populations in LN macrophage subpopulations after LPS and SC MSC injection. Data expressed as mean ± SEM; n = 5 mice per group, representative of 2 independent experiments. One-way ANOVA, Tukey’s multiple comparison, comparing UNT, LPS and MSC dLNs; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant

Fig. 4

SC MSC injection expands activated Tregs in the LN. (A) CD45⁺Ly6G⁺SSC-A^hi neutrophils, Ly6C^lo/hiCD11b⁺ monocytes, CD11c⁺MHCII⁺ DCs and CD11b⁺MerTk⁺ macrophages were purified from the LNs 24 h after SC MSC injection, and then added to CTV-labelled, anti-CD3/anti-CD28 activated splenic CD4⁺CD25⁻ T cells at 1:5 ratio for 72 h. (B) Flow cytometric profile of CTV dilution (indicating T cell proliferation) in the presence of innate immune cells purified from LN of untreated mice (UNT LN) or LNs from SC MSC-injected mice (MSC LN). (C) Division index of T cell proliferation. Data expressed as mean ± SEM; n = 3 mice per group. (D) Representative flow cytometry plot showing Treg gating. (E) Relative change in Treg proportion in the presence of LN neutrophils, monocytes, DCs or macrophages after standardising to T cells alone group). Data expressed as mean ± SEM; n = 3 mice per group. (F) Draining (dLN; white bar) and non-draining LNs (ndLN; shaded bar) were harvested from mice that received SC injection of viable or apoptotic MSCs into the inflamed hock. Flow cytometry plots showing viability of MSCs treated with DMSO (mock) or BH3 mimetics prior to SC injection. (G) Treg cellularity in the dLN (white bars) and ndLNs (shaded bars) following LPS and SC MSC injection. (H) Changes in the PD-1 expression on Tregs after SC MSC injection, and Pearson correlation analysis of Treg PD-1 expression level and Treg proportion in the dLNs from LPS and MSC groups (Pearson r=-0.861, p = 0.00137). (I) CD44 expression on activated Tregs, and changes in CD44^hi Tregs in dLNs and ndLNs after SC MSC injection. (J) Changes in PD-1 expression on CD44^hiTregs after SC MSC injection, and Pearson correlation analysis of CD44^hi Treg PD-1 expression level and Treg proportion in the dLNs from LPS and MSC groups (Pearson r=-0.883, p = 0.001). Data expressed as mean ± SEM; n = 5 mice per group. One-way ANOVA, Tukey’s multiple comparison, comparing UNT, LPS and MSC dLNs; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (K) Treg cellularity in dLNs and ndLNs following SC injection with ApoMSC. (L) Changes in PD-1 expression on Tregs after SC injection with ApoMSC, and Pearson correlation analysis of Treg PD-1 expression level and Treg proportion in the dLNs from LPS and ApoMSC groups (Pearson r=-0.239, p = 0.392). Data expressed as mean ± SEM; n = 5–10 mice per group. One-way ANOVA, Tukey’s multiple comparison, comparing UNT, LPS and ApoMSC dLNs; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant

Fig. 5

SC MSC injection inhibits TNF production by LN neutrophils in response to LPS re-exposure. (A) C57BL/6 mice were SC-injected with 30 ng LPS in 50 µl PBS in the left hock, and 1 h later 1 × 10⁶ human adipose MSCs in the same hock. The popliteal LN draining the LPS-injected hock was harvested 4 h after LPS injection (i.e. 3 h after MSC treatment) and restimulated with 10 µg/ml LPS for 4 h in vitro. Intracellular TNF production was used as an indicator of the pro-inflammatory response to the LPS rechallenge. (B) Flow cytometry plots showing the gating strategies for identifying the TNF-producing (TNF⁺) populations after in vitro restimulation of bulk LN cells with LPS. (C) Representative flow cytometry plots showing the gating for TNF⁺ neutrophils. (D) Number of TNF⁺ neutrophils after LPS restimulation of LN cells from mice that received SC injection of viable MSCs (left panel) or ApoMSC (right panel). Data expressed as mean ± SEM; n = 5–10 mice per group from 2 independent experiments. One-way ANOVA, Tukey’s multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. E) Representative flow cytometry plots showing the gating for TNF⁺ monocytes. (F) Number of TNF⁺ monocytes after LPS restimulation of LN from mice that received SC injection of viable MSCs (left panel) or ApoMSC (right panel). Data expressed as mean ± SEM; n = 5–10 mice per group from 2 independent experiments. One-way ANOVA, Tukey’s multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (G) Cellularity and expression of MHCII, CD86 and PD-L1 on neutrophils in the dLN after SC MSC injection. Data expressed as mean ± SEM; n = 5 mice per group. One-way ANOVA, Tukey’s multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant

Fig. 6

Contralateral SC MSC injection does not inhibit LN neutrophil TNF response to LPS re-exposure. (A) C57BL/6 mice were SC-injected with 30 ng LPS in 50 µl PBS in the left hock, and 4 h later the skin around the LPS injection site (LPS inflamed) and on the contralateral hock (LPS non-inflamed) was excised and homogenised for cytokine analysis. Data expressed as mean± SEM; n = 10 mice per group from 2 independent experiments. One-way ANOVA, Tukey’s multiple comparison, * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (B) Bioluminescent imaging of mice that received SC injection of 30 ng LPS in 50 µl PBS in the left hock, and 1 h later 1 × 10⁶ luciferase-expressing MSCs in the same hock, or (C) in the non-inflamed hock on the contralateral side. (D) Bar graphs represent changes in radiance over time. n = 3 mice per group from one experiment. (E) C57BL/6 mice were SC-injected with 30 ng LPS in 50 µl PBS in the left hock. 1 h after LPS injection, mice received a SC injection of 1 × 10⁶ human adipose MSCs (MSC-c) or control PBS treatment (PBS-c) in the contralateral (non-inflamed) hock. The popliteal LNs draining the LPS-injected and the contralateral skin were harvested 4 h after LPS injection (i.e. 3 h after MSC-c/PBS-c treatment) and restimulated with 10 µg/ml LPS for 4 h. Intracellular TNF production was used as an indicator of the pro-inflammatory response to the LPS rechallenge. (F) Number of TNF⁺ neutrophils after LPS restimulation of LN cells from mice that received SC injection of MSC or control PBS on the contralateral hock. Data expressed as mean ± SEM; n = 5 mice per group. One-way ANOVA, Dunnett’s multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant. (G) Number of TNF⁺ monocytes after LPS restimulation of LN cells from mice that received SC injection of MSC or control PBS on the contralateral hock. Data expressed as mean ± SEM; n = 5 mice per group. One-way ANOVA, Dunnett’s multiple comparison; * p < 0.05, ** p < 0.01, *** p < 0.001, ns, not significant.