MSCs administered via the IV route undergo apoptosis in the lung, a process shown to be necessary for inducing anti-inflammatory effects 8. As expected, IV-injected MSCs were detected in the lung immediately after injection, and a rapid decay of their bioluminescent signals was observed within 1 day, with complete clearance on day 3 (Fig. 1B). IP-injected MSCs remained in the peritoneum around the injection site and the signals persisted for a slightly longer period of time than IV-injected MSCs, with complete clearance on day 4 (Fig. 1B). Compared to the IV or IP injection groups, MSCs administered via the SC route into the hock displayed the longest in vivo dwell time, with complete clearance on day 5 (Fig. 1B). SC-injected MSCs remained locally at the injection site (hock), without evidence of migration away from the injection site.
SC MSC injection into inflamed skin expands immunoregulatory IL-10+ macrophages in the draining LN
In inflammation and other disease states, immune responses are initiated and regulated in secondary lymphoid organs such as LNs. Studies characterizing the LN response to an acute inflammatory stimulus have commonly examined the popliteal lymph node draining the footpad where the inflammatory stimulus was injected 22. The popliteal lymph nodes in mice are very small but easily located and separated from the skin and fascia 23. Therefore, having confirmed that SC-injected MSCs remained at the injection site over a longer duration than IV- or IP-injected MSCs, we next established a localised acute inflammation model to provide an inflammatory microenvironment for SC-injected MSCs that would enable us to analyse the draining popliteal LN response.
TLR4 signalling pathway has been shown to be involved in establishing inflammatory hyperalgesia via the induction of pro-inflammatory cytokines such as TNF and IL-1β 24. In a previous study, 100ng of LPS (a TLR4 agonist) induced pro-inflammatory cytokines in the paw skin and inflammatory pain in the paw 24. Due to institutional ethical constraints around animal welfare, we injected LPS into the hock, which is a non-weight bearing structure that drains to the same lymph node as the footpad, as a humane alternative to footpad injections 25. We found 30ng LPS injected into the hock was sufficient in inducing an acute inflammatory response in the skin, indicated by TNF, IL-1β and MCP-1 production, which peaked at 4 h post-LPS injection and resolved by 24 h (Fig. 2A), with concurrent expansion in total cellularity of the draining popliteal LN, increases in neutrophil and monocyte cell numbers from 4 h to 24 h (Fig. 2B) and elevation in their pro-inflammatory gene expression (TNF, IL-1β, IL-6 and MCP-1) (Fig. 2C).
Adipose tissue-derived MSCs were then delivered via SC injection into the inflamed tissue for in vivo priming with TNF, IL-1β and MCP-1 (which are cross-reactive between mouse and human cells 26) and the popliteal LN analysed at 24 h (Fig. 3A, B) when the SC-injected MSCs were still detectable (Fig. 1B). Although neutrophils and monocytes in the LN expanded in number in response to LPS injection and were the main producers of proinflammatory cytokines in this model (Fig. 2C), SC MSC injection did not affect the cellularity of these populations, nor the number of DCs or T cells (Fig. 3C). There was, however, a significant increase in B cells in the LN following SC injection of MSCs into the inflamed tissue (Fig. 3C, white bar), which was not found in the non-draining popliteal LN (LN from the non-inflamed hock; Fig. 3C, shaded bar). Regulatory B cells (Bregs) have been reported to attenuate inflammation and are characterised by their ability to produce IL-10 27, 28, 29, 30. We examined IL-10 production in B cells and found no increase in IL-10+ B cells (Fig. 3C), suggesting that SC MSC injection expanded the bulk B cell population rather than a specific regulatory B cell subtype.
In the lymph node, resident macrophages play crucial roles in immune regulation. Subcapsular sinus macrophages (SSM) act as gatekeepers by capturing foreign antigens in the lymph, whilst medullary sinus macrophages (MSM), medullary cord macrophages (MCM), tingible body macrophages and T cell zone macrophages (TZM) actively clear apoptotic cell debris, contributing to tissue homeostasis 19. Following SC MSC injection, there was also an increase in MerTK+ SSM and TZM, but not F4/80+ MSM and MCM (Fig. 3D, white bar). None of these cellularity changes in response to LPS and MSC injection was observed in the non-draining popliteal LN (Fig. 3D, shaded bar).
LN-resident macrophages possess immunoregulatory function that protect against pathogenic infection and inflammatory insults 31, 32, and MerTk signalling in macrophages has been linked to anti-inflammatory outcomes 33, 34. IL-10 has also been shown to shift macrophages toward an immunoregulatory and tolerogenic phenotype 35. We therefore investigated whether the expansion of MerTK+ LN macrophages after SC MSC injection is associated with anti-inflammatory IL-10 production (Fig. 3E). The number of IL-10-producing macrophages remained unchanged in LPS mice, but SC MSC injection increased the number of IL-10-producing SSM and TZM in the draining popliteal LN (Fig. 3F, white bar). This increase was not observed in F4/80+ macrophages (Fig. 3F). IL-10+ macrophages also remained unchanged in the non-draining LN (Fig. 3F, shaded bar). Thus, SC MSC injection induced an increase in IL-10-producing MerTK+ macrophages but not F4/80+ macrophages in the draining LN.
SC MSC injection into inflamed skin expands activated Tregs with decreased PD-1 expression in the draining LN
Following IV administration of MSCs, MSC-primed monocytes/macrophages have been shown to enhance the differentiation of T cells to Tregs 7, 36, 37, which have crucial immunoregulatory function in the LN 38, 39, 40. The increase in IL-10+ MerTK+ macrophages in the LN after SC MSC injection prompted us to examine the immunomodulatory capacity of macrophages following SC MSC injection. MerTK+ macrophages (SSM and TZM), neutrophils, monocytes and DCs were isolated from the LN of MSC-treated mice and co-cultured with CTV-labelled CD4+CD25− T cells that were activated with anti-CD3/anti-CD28 (Fig. 4A). Activated T cells underwent proliferation, as indicated by CTV dilution (Fig. 4B). Neither macrophages, neutrophils, monocytes nor DCs suppressed T cell proliferation (Fig. 4C). However, analysis of Foxp3+ Tregs in the co-culture (Fig. 4D) revealed that macrophages from the LN of SC MSC-injected mice increased the proportion of Tregs, compared to macrophages from untreated mice (Fig. 4E). The expansion of Tregs was not observed with neutrophils, monocytes or DCs from the same LN of SC MSC-injected mice (Fig. 4E). Thus, MerTK+ macrophages from the LN of mice that received SC MSC injection exhibited an enhanced capacity to differentiate and/or expand Tregs.
As MerTK+ macrophages from the LN of SC MSC-injected mice displayed increased IL-10 production and enhanced capacity for Treg expansion, we performed a more detailed analysis of the Treg population within the LN (Fig. 4F). There was a pronounced increase in Foxp3+ Tregs in the draining popliteal LN of MSC-treated mice (Fig. 4G). Strikingly, Tregs from SC MSC-injected mice displayed decreased PD-1 expression (Fig. 4H). PD-1 expression on Tregs has been shown to be inversely proportional to their suppressive activity 41, 42. A recent study showed that PD-1 on Tregs inhibits Treg activation and suppressive activity, as PD-1-deficient Tregs are more immunosuppressive 43. PD-1–deficient Tregs exhibit an activated phenotype (CD44hi CD62Llo) 43. In line with this, there was an increase in activated CD44hi Tregs (Fig. 4I), which displayed decreased PD-1 expression levels (Fig. 4J), in the LN of SC MSC-injected mice. No significant difference in Tregs was observed in the non-draining LN of SC MSC-injected mice. Furthermore, no changes in the number of Tregs (Fig. 4K), or their PD-1 expression (Fig. 4L), were observed in the LN following SC injection of apoptotic MSCs (ApoMSC). Thus, SC injection of viable MSCs but not ApoMSCs increased the number of activated CD44hi Tregs with decreased PD-1 expression in the dLN.
SC MSC injection into inflamed skin inhibits the TNF response of LN neutrophils to LPS rechallenge
Having found that SC MSC injection promoted immunoregulatory cells in the LN draining the inflamed tissue, we next examined whether there was a suppression of the inflammatory response by analysing the production of pro-inflammatory TNF by LN cells following SC MSC injection. As MSCs were detectable up to 5 days after SC injection (Fig. 1B), a group of mice was injected with ApoMSCs as comparison, and the popliteal LN draining the inflamed tissue was analysed 4 h after LPS injection (Fig. 5A, B). Neutrophils were robust producers of TNF in response to LPS restimulation, with neutrophils accounting for ~ 40% of TNF-producing cells in the draining LN (Fig. 5C). Over 60% of neutrophils stained positive for intracellular TNF production (Fig. 5D). The popliteal LN from mice injected with viable MSCs showed a reduction in TNF-producing neutrophils upon restimulation with LPS (Fig. 5E, left panel). The reduction in TNF+ neutrophils was not observed in LN from mice that received ApoMSC (Fig. 5E, right panel). There were no significant changes in TNF+ monocytes with either viable MSC or ApoMSC injection (Fig. 5F, G). Further examination of neutrophils in the LN showed that whilst LPS injection increased their cell number and MHC class II expression, there were no significant changes with SC MSC injection (Fig. 5H). Thus, SC MSC injection decreased the TNF response of LN neutrophils to LPS, which required MSCs to be viable at the time of injection. The reduction in TNF was not due to a decrease in the number, or activation, of neutrophils in the LN.
Direct MSC injection into the inflamed skin is necessary for immunoregulatory effects in the LN
The immunomodulatory effects induced by SC MSC injection were observed in the LN draining the inflamed hock, but not in the non-draining LN. We therefore investigated whether SC MSC injection into the non-inflamed hock would induce the same effects. Using the above LPS-induced inflammation model, we first confirmed that LPS injection into the hock led to an increase in pro-inflammatory cytokines TNF, IL-1b, IL-6 and MCP-1 in the skin from the inflamed hock and did not induce any cytokine changes on the contralateral side (non-inflamed hock) (Fig. 6A). We then repeated the bioluminescence imaging to determine whether local inflammation affects the in vivo persistence of SC-injected MSCs. Fluc-GFP+ MSCs were injected via the SC route into the inflamed skin area (Fig. 6B) or the non-inflamed contralateral hock (Fig. 6C) 1 h post-LPS injection. Mice were imaged 10 min after SC MSC injection, 3 h later and then daily. Local inflammation did not increase the bioluminescent signals of fluc-GFP+ MSCs, neither did inflammation at a distance from MSC injection drive cell migration beyond the injection site (Fig. 6B, C). The bioluminescent signals lasted around 5 days, with a slight increase in bioluminescence signal during the first 2 days (Fig. 6D). Direct imaging of the popliteal LNs did not show drainage of significant numbers of MSCs to the local popliteal LN (data not shown). Although unlikely, we cannot rule out the potential dissemination of small numbers of MSCs, which were below the detection range, to other sites of the body.
We next investigated whether contralateral SC MSC injection into the non-inflamed hock could remotely regulate the inflammatory response to LPS within the draining LN (Fig. 6E). Analysis of the popliteal LN draining the inflamed hock 4 h after LPS injection showed that neither contralateral MSC injection nor PBS control treatment had an effect on the number of TNF-producing neutrophils or monocytes in response to LPS restimulation (Fig. 6F, G; inflamed). There was also no change in the number of TNF+ neutrophils or monocytes in the popliteal LN from the non-inflamed hock in any of the treatment groups (Fig. 7F, G; non-inflamed). Thus, in contrast to direct MSC injection into inflamed tissue, contralateral MSC injection did not affect the LN inflammatory response to LPS. This finding indicates that ‘priming’ of SC-injected MSCs by inflammatory cytokines in the local milieu is important for their immunoregulatory effects in the LN.
Taken together, our data demonstrate that SC injection of MSCs into inflamed tissue induced an increase in immunoregulatory cells in the tissue-draining LN and reduced the responsiveness of neutrophils to subsequent inflammatory challenge. In contrast to IV delivery of MSCs, the immunoregulatory effects of SC MSC injection on the local draining LN are not coupled with MSC apoptosis.