Mucosally transplanted mesenchymal stem cells stimulate intestinal healing by promoting angiogenesis

Mesenchymal stem cell (MSC) therapy is an emerging field of regenerative medicine; however, it is often unclear how these cells mediate repair. Here, we investigated the use of MSCs in the treatment of intestinal disease and modeled abnormal repair by creating focal wounds in the colonic mucosa of prostaglandin-deficient mice. These wounds developed into ulcers that infiltrated the outer intestinal wall. We determined that penetrating ulcer formation in this model resulted from increased hypoxia and smooth muscle wall necrosis. Prostaglandin I(2) (PGI(2)) stimulated VEGF-dependent angiogenesis to prevent penetrating ulcers. Treatment of mucosally injured WT mice with a VEGFR inhibitor resulted in the development of penetrating ulcers, further demonstrating that VEGF is critical for mucosal repair. We next used this model to address the role of transplanted colonic MSCs (cMSCs) in intestinal repair. Compared with intravenously injected cMSCs, mucosally injected cMSCs more effectively prevented the development of penetrating ulcers, as they were more efficiently recruited to colonic wounds. Importantly, mucosally injected cMSCs stimulated angiogenesis in a VEGF-dependent manner. Together, our results reveal that penetrating ulcer formation results from a reduction of local angiogenesis and targeted injection of MSCs can optimize transplantation therapy. Moreover, local MSC injection has potential for treating diseases with features of abnormal angiogenesis and repair

Type I Interferons Link Viral Infection to Enhanced Epithelial Turnover and Repair

The host immune system functions constantly to maintain chronic commensal and pathogenic organisms in check. The consequences of these immune responses on host physiology are as yet unexplored, and may have long-term implications in health and disease. We show that chronic viral infection increases epithelial turnover in multiple tissues, and the antiviral cytokines type I interferons (IFNs) mediate this response. Using a murine model with persistently elevated type I IFNs in the absence of exogenous viral infection, the Irgm1−/− mouse, we demonstrate that type I IFNs act through nonepithelial cells, including macrophages, to promote increased epithelial turnover and wound repair. Downstream of type I IFN signaling, the highly related IFN-stimulated genes Apolipoprotein L9a and b activate epithelial proliferation through ERK activation. Our findings demonstrate that the host immune response to chronic viral infection has systemic effects on epithelial turnover through a myeloid-epithelial circuit

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

IL-6 expression was increased in human colons at sites of perforation.

<p>Tissue that was surgically resected from patients who suffered large bowel perforation was evaluated for IL-6 expression by <i>in situ</i> hybridization. Eleven cases were evaluated (8 males with trauma due to gun-shot wounds, ages 16–33; 1 female surgical trauma, age 45; and 2 females with diverticulitis, ages 72 and 79). (A) Representative staining from a patient with diverticulitis is shown at 20X and 100X, respectively. Bars = 100 µm. Arrows indicate IL-6+ cells with lymphocyte morphology. (B) Four high-powered fields with well-oriented crypts were evaluated for IL-6+ cells in the epithelial layer at the site of perforation and at the distal resection margin. The average ratio of IL-6+ cells in the perforation versus distal site ± SEM was shown. An unpaired student's t-test was used for statistical analysis; *, <i>P</i> = 0.02.</p

Inhibition of IL-6 resulted in more severe colitis and inhibition of intestinal epithelial proliferation.

<p>Colitis was induced in <i>dnKO</i> and <i>IL-10rb^+/-</i> littermate controls by co-housing. On day zero and three times weekly, mice were injected intraperitoneally with 500 µg of either anti-IL-6 mAb or control IgG mAb. Two independent experiments were performed with 8-9 mice/group. (A) Plot of the average percent of starting weight ± SEM shown for indicated groups of mice. Mice were weighed every three days. (B) Representative H+E stained sections of descending colons at day 9 post co-housing. Bars = 500 µm. Black dotted lines outline remaining crypts in the <i>dnKO</i> anti-IL-6 mAb treated mouse histology. (C) Graph of the average number of descending colonic crypts per high-powered field ± SEM. (D) At day 9 post-co-housing, mice were injected with BrdU one hour before sacrifice. Representative colonic sections stained with mAb to BrdU and detected with fluorescently conjugated antibodies were shown. The white dotted lines delineate crypts. Bars = 100 µm. (E) Graph of the average number ± SEM of BrdU positive cells per crypt. (F) Graph of the average ± SEM number of apoptotic bodies/crypt. One-way analysis of variance: (A) F = 3.5, <i>P</i><0.05 (for day 9 weights); (C) F = 57.36, <i>P</i><0.0001; (E) F = 17.92, <i>P</i><0.0001; (F) F = 10.87, <i>P</i><0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.</p

IL-6 promoted intestinal epithelial proliferation in wound biopsy model.

<p>(A) WT mice were biopsy injured in the distal colon. Plot of the relative levels of IL-6 mRNA expression in the wound bed (relative to uninjured tissue) for various times after injury. N = 2–3 WT mice with a total of 4-6 wounds/time point. Data were shown as average ± SEM. One-way analysis of variance: F = 5.68, <i>P</i><0.01 (B) Cartoon depicting the microanatomy of a wound at day six post-biopsy; AC  =  adjacent crypts (green area); WC  =  wound channels (blue area); WAE  =  wound-associated epithelium overlying the wound bed (pink area). (C) Colonic sections of wounds from <i>IL-6^+/−</i> and <i>IL-6^-/-</i> mice at day six post-injury stained with mAb to BrdU (labels S-phase cells, red), mAb to β-catenin (labels epithelium, green), and bis-benzimide (nuclei, blue). Bars = 500 µm. (D) Quantification of the number of BrdU positive cells/wound adjacent crypts. Data were graphed as average ± SEM. One way analysis of variance: F = 10.5, p<0.0001. Means with different letters are significantly different by Bonferroni's multiple comparison test.</p

Intraepithelial lymphocytes were a source of IL-6 early after injury.

<p>(A) Biopsy of the colon mucosa was performed in WT mice to create small wounds. IL-6 expression in the wound bed and adjacent tissue was evaluated by <i>in situ</i> hybridization one day after biopsy. Representative images were shown. Bars = 200 µm. Colored bars above wound images indicate areas of the wound bed as depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0114195#pone-0114195-g004" target="_blank">Figure 4B</a>. (B) Co-localization by immunofluorescence was performed for IL-6 (red), CD3ε (green), and bis-benzimide (blue) on colon tissue from <i>dnKO</i> mice at day 6 after co-housing. Representative staining was shown at 63X. Bar = 200 µm. (C) Epithelial cells were harvested from <i>dnKO</i> mice on day 6 after co-housing, stained for T cell markers and IL-6, and assessed by flow cytometry. Representative dot plots were shown. (D, E) CD3+ CD4- CD8- IELs were harvested from WT mice and stimulated <i>ex vivo</i> with 10 ng/ml PMA and 1 µg/ml ionamycin for 5 hours. (D) RNA was collected and evaluated by qRT-PCR for IL-6 expression. (E) Culture supernatants were harvested and evaluated for secreted IL-6 by electrochemilluminescence. Data were shown as the average IL-6 expression or protein ± SEM. A paired student's t-test was used to determine significance; *, <i>P</i><0.05.</p

IL-6 stimulates intestinal epithelial proliferation and repair after injury.

IL-6 is a pleiotropic cytokine often associated with inflammation. Inhibition of this pathway has led to successful treatment of rheumatoid arthritis, but one unforeseen potential complication of anti-IL-6 therapy is bowel perforation. Within the intestine, IL-6 has been shown to prevent epithelial apoptosis during prolonged inflammation. The role of IL-6 in the intestine during an initial inflammatory insult is unknown. Here, we evaluate the role of IL-6 at the onset of an inflammatory injury. Using two murine models of bowel injury - wound by biopsy and bacterial triggered colitis - we demonstrated that IL-6 is induced soon after injury by multiple cell types including intraepithelial lymphocytes. Inhibition of IL-6 resulted in impaired wound healing due to decreased epithelial proliferation. Using intestinal tissue obtained from patients who underwent surgical resection of the colon due to traumatic perforation, we observed cells with detectable IL-6 within the area of perforation and not at distant sites. Our data demonstrate the important role of IL-6 produced in part by intraepithelial lymphocytes at the onset of an inflammatory injury for epithelial proliferation and wound repair