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, 9 (4), e95720

The Role of Indoleamine 2,3 Dioxygenase in Beneficial Effects of Stem Cells in Hind Limb Ischemia Reperfusion Injury


The Role of Indoleamine 2,3 Dioxygenase in Beneficial Effects of Stem Cells in Hind Limb Ischemia Reperfusion Injury

Mohamad Masoumy et al. PLoS One.


Ischemia-Reperfusion (IR) injury of limb remains a significant clinical problem causing secondary complications and restricting clinical recovery, despite rapid restoration of blood flow and successful surgery. In an attempt to further improve post ischemic tissue repair, we investigated the effect of a local administration of bone marrow derived stem cells (BMDSCs) in the presence or absence of immune-regulatory enzyme, IDO, in a murine model. A whole limb warm ischemia-reperfusion model was developed using IDO sufficient (WT) and deficient (KO) mice with C57/BL6 background. Twenty-four hours after injury, 5 × 105 cells (5×105 cells/200 µL of PBS solution) BMDSCs (Sca1 + cells) were injected intramuscularly while the control group received just the vehicle buffer (PBS). Forty-eight to seventy-two hours after limb BMDSC injection, recovery status including the ratio of intrinsic paw function between affected and normal paws, general mobility, and inflammatory responses were measured using video micrometery, flow cytometry, and immunohistochemistry techniques. Additionally, MRI/MRA studies were performed to further study the inflammatory response between groups and to confirm reconstitution of blood flow after ischemia. For the first time, our data, showed that IDO may potentially represent a partial role in triggering the beneficial effects of BMDSCs in faster recovery and protection against structural changes and cellular damage in a hind limb IR injury setting (P = 0.00058).

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.


Figure 1
Figure 1. Treatment Effect on Toe Spread Ratio Averages (48–72 hours after treatment).
The outcome of stem cell (SC) therapy indicates that IDO may improve recovery. IDO-KO mice treated with SC demonstrated an accelerated recovery compared with IDO-KO treated with PBS (p-value <0.05). However, the WT mice treated with SC showed the greatest recovery of intrinsic paw function when expressed as a ratio comparing it to the non-injured paw (p-value = 0.027). Functional recovery from ischemia-reperfusion (IR) injury in the different treatment groups was measured, using a modified version of walking track analysis. For each subject, toe spread was measured in the IR limb (Ti) and control contralateral limb (Tc). The ratio of the toe spread in the injured limb (Ti) to the control limb (Tc) was then calculated by Ti/Tc. A ratio of 1 indicates 100% recovery or equal width and thus normal intrinsic function. When comparing the WT group treated with stem cells to those treated with PBS, a 45% increase in recovery was seen demonstrating the efficacy of stem cell therapy alone in the presence of an environment where IDO expression is present.
Figure 2
Figure 2. T2 weighted images.
a) Representative T2 weighted imaging cuts where the enhancement of the soft tissue can be observed within the injured legs and compared to the non-injured legs and, b) The average ratio of enhancement measured between the injured leg over the non-injured leg are shown in this representative graph for each of the 4 groups.
Figure 3
Figure 3. BMDScs can enhance IDO and regulatory T cells while reducing inflammatory cytokines in the hind limb IR injury.
Immunohistochemical analysis of paraffin embedded tissues from murine model with IRI of hind limb showed that treating the animals with BMDSCs in an IDO sufficient microenvironment first: increased IDO and FOXP3 expression (panels A and B, red arrows), while decreased the inflammatory cytokines, IL-17 and IL-23 (panels C and D). Anti inflammatory cytokine, IL-10, was increased as demonstrated in panel E. All together, these analysis suggest a potential therapeutic role for BMDSCs, re-enforced by possible IDO dependent mechanisms. All pictures are 400X magnification.
Figure 4
Figure 4. Regulatory effects of SCs may be partially associated with IDO presence in tissue microenvironment.
Dot Plots are representatives of flow cytometric analysis of live single cell preparations from murine hind limb tissues with IRI showed that in the presence of IDO, injection of BMDSCs decreased CD3+ cells (T cells), IL-17 and IL-23 expression (IL-23 is based on gating of IL-17+ cells). Further, applying BMDCs could increase both CD3+ FOXP3+ cells (Tregs, gated on CD3+ cells) and IL-10 expression in an IDO sufficient microenvironment. Bargraphs on the right side of each group are reflecting the accumulative analysis of five animal per experimental group. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple-comparison post- hoc test. * = p<0.05, ** = p<0.01, *** = p<0.001.
Figure 5
Figure 5. SCs treatment reduces cell death in an IDO dependent manner.
Flow cytometry analysis showed (5a) that stem Cells reduce cell death (apoptosis/necrosis) with their most profound effects in the presence of IDO. Further, flow cytometric analysis demonstrated that a majority of reduction in cell death occurred among parenchymal cells (CD45 negative) and not infiltrated leukocytes (CD45+), indicating the beneficial impact of SCs in reducing cell death and necrotic responses, leading to less inflammation, more protection and faster recovery. Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple-comparison post- hoc test. * = p<0.05, ** = p<0.01, *** = p<0.001.

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Institutional Seed Money. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.