Immunology

Permanent URI for this collectionhttps://hdl.handle.net/20.500.12503/21658

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Atrophied thymus produces altered repertoire of tTregs with potential to break the balance of peripheral tolerance
(2018-03-14) Oh, Jiyoung; Wang, Weikan; Su, Dong-Ming; Thomas, Rachel
Purpose: We have previously shown that in the age-related atrophied thymus, there is an increased ratio of thymic T regulatory (tTreg) cells to thymic T conventional (tTcon) cells, suggesting that the aged thymus has an enhanced tTreg generation. It is also known that in the periphery of aged mice and humans, there is an accumulation of peripheral Tregs. We raise the following question: why is the increased Treg population unable to suppress self-reactivity in the elderly? Methods: We utilized a sub-lethally irradiated chimeric mouse model in which OT-II TCR transgenic bone-marrow cells were transplanted into RIP-mOVA host mice. Therefore, OVA serves as a mock self-antigen, and because this transgene is driven by the rat insulin promotor, the pancreas of these mice expresses OVA. Further, the mOVA mice also carry a FoxN1-floxed gene, which can be conditionally knocked-out via CreERT transgene activation by injection(s) with tamoxifen to induce thymic atrophy. Results: We observed that chimera mice with induced thymic atrophy show total Tregs (termed pan-Tregs) were increased but OT-II specific Tregs were slightly decreased in the spleen. Additionally, there is a dramatic decrease in the OTII-specific Tregs found in the pancreas, and pancreatic atrophy was observed in these mice compared to those with normal thymus. Conclusions: Our initial results suggest that thymic atrophy leads to a biased tTreg generation that manifests in decreased self-tissue specific-Tregs. Therefore, although pan-Tregs may accumulate and be enhanced with age, tissue-specific peripheral tolerance may be poised to fail.
Extracellular Superoxide Dismutase Hinders Effective Containment of Listeria monocytogenes by Neutrophils
(2018-03-14) Berg, Rance E.; Okunnu, Busola
Purpose: Increased activity of Extracellular Superoxide Dismutase (ecSOD), an enzyme widely regarded as having protective functions during ROS induced inflammation, has been shown to be detrimental to host survival during infection with the intracellular bacteria, Listeria monocytogenes (Lm). Although, we have also demonstrated that neutrophils are essential for protection during Lm infection, a higher percentage of neutrophils are present in mice with high ecSOD activity during Lm infection. However, these mice are still more susceptible to infection in comparison to mice that lack ecSOD activity. These paradoxical findings led to the objective to better understand how ecSOD activity modulates the protective functions of neutrophils during Lm infection. Materials and Methods: For these studies, ecSOD congenic mice: ecSOD HI mice with high ecSOD activity, ecSOD WT mice with normal ecSOD activity, and ecSOD KO mice with no ecSOD activity were utilized. To determine phagosomal containment, we made use of flow cytometry and a strain of Lm, actA:LMGFP, which only fluoresces GFP when the bacteria escapes out of the phagosome into the cytosol. Results: A higher percentage of neutrophils from the ecSOD KO mice took up Lm in comparison to the HI neutrophils. Correlated with this was also a higher percentage of ecSOD KO neutrophils allowing for phagosomal escape in comparison to the ecSOD HI neutrophils. Analysis of the mean fluorescence intensity (MFI) showed that although there were more bacteria present in the ecSOD KO neutrophils, the amount of escaped bacteria was comparable to that in the ecSOD HI neutrophils. Treatment of the ecSOD KO neutrophils with IFN-g, an activator of neutrophils, also led to better phagosomal containment. Though the IFN-g treated neutrophils took up more bacteria, there was no difference in the amount of escaped bacteria in comparison to the non-treated cells. Conclusion: EcSOD activity hinders the ability of neutrophils to keep Lm contained in the phagosome which prevents effective bacterial killing. Additionally, neutrophil activation with IFN-g also makes the cells more effective at bacterial containment. However, the effect of ecSOD activity in conjunction with IFN-g is yet to be determined. Future studies on how ecSOD affects bacterial killing and other functions downstream of phagosomal escape will aid in a better understanding of how ROS can modulate neutrophil function during intracellular bacterial infections.
Atrophied thymus can serve as a tumor reservoir for harboring melanoma cells
(2018-03-14) Kuriatnikov, Denis; Su, Dong-Ming; Sizova, Olga
Purpose: Tumor metastatic relapse is responsible for main cancer associated mortality and potentially arises from the undetectable minimal number of tumor cells, which are able to resist radio-chemotherapy at a dormant state hiding in certain organs (termed: tumor reservoirs). The largest T-lymphoid organ, the thymus, has been suggested as this kind of pre-metastatic tumor reservoir for B-lymphoma cells. It remains unknown whether the thymus is able to harbor non-lymphoid solid tumor cells, why chemotherapy cannot thoroughly eliminate cancer cells in the thymus, and what the state of thymic occult cancer cells is during chemotherapy. Methods: With melanoma inoculated and genotoxic doxorubicin (Doxo) treated mouse model, we determined that the thymus, particularly the atrophied thymus, was able to harbor blood stream-circulating melanoma cells. Using specific in vivo + in vitro technique, where thymuses of doxorubicin- or PBS-treated mice are co-cultured with doxorubicin-treated melanoma cells in trans-well system we want to provide the insight of the changes in the status of cancer cells. Results: We found that chemotherapy-resulted DNA-damage response triggered p53 activation in non-malignant thymic cells, which in turn resulted in thymocyte death and thymic epithelial cell senescence to develop an inflammatory thymic microenvironment. Co-culture of PBS- or Doxo- treated thymus with Doxo-treated melanoma cells provides evidence that chemotherapy-altered inflammatory thymic microenvironment protects cancer cells from apoptosis via induction of dormancy. Conclusion: Therefore, the thymus, which becomes a pre-metastatic reservoir for non-lymphoid solid tumor cells under chemotherapy, should be a novel target in antitumor therapy for considering and preventing from tumor metastatic relapse.
Lymphatic pump technique mobilizes lymph that inhibits the inflammatory response of macrophages in vitro
(2018-03-14) Hodge, Lisa; Castillo, Rudy A.
Purpose: The lymphatic system maintains tissue fluid homeostasis by returning excess fluid, known as lymph, into circulation. Osteopathic physicians recognize the importance of the lymphatic system and have designed a set of lymphatic pump techniques (LPT) that enhance the flow of lymph. LPT has been used clinically to treatment respiratory tract disease, infection, and edema. LPT has also been reported to enhance mesenteric and thoracic lymphatic flow, the concentration of leukocytes, and the flux of inflammatory mediators in lymph of dogs. We propose LPT may act as an adjunctive therapy by mobilizing lymph-borne factors into circulation that protect tissues during inflammation. In this study, we propose that lymph mobilized by LPT suppresses macrophage activation in vitro. Methods: To test this hypothesis, under anesthesia, the thoracic ducts of six mongrel dogs were cannulated and thoracic duct lymph (TDL) was collected before (baseline), during, and after (recovery) LPT. TDL supernatant was collected by centrifugation and frozen. Total protein was measured in TDL using the Bradford method. Murine RAW 264.7 macrophages were cultured with baseline, LPT, or recovery TDL at 5% total volume per well with or without lipopolysaccharide (LPS) for 24 hours at 37°C and 5% CO2. After culture, cell-free supernatants were assayed for nitrite (NO2-), tumor necrosis factor-alpha (TNF-alpha), and interleukin-10 (IL-10). Macrophage viability was measured using flow cytometry and markers, annexin V and propidium iodide. Results: LPT transiently increased TDL flow and protein flux (10-fold). Baseline, LPT, or recovery TDL did not increase the production of NO2-, TNF-alpha, IL-10 or alter macrophage viability. When macrophages were activated with LPS, the addition of baseline, LPT, or recovery TDL decreased the production of NO2- (2-fold), TNF-alpha (5-fold) and IL-10 (3-fold) compared to LPS. There were no significant (p [greater than] 0.05) differences in the production of NO2-, TNF-alpha, and IL-10 by macrophages cultured with baseline, LPT, or recovery TDL and LPS. Conclusions: Our data suggests that lymph contains biological factor(s) that suppress macrophage activation without altering cell viability. The redistribution of protective lymph during LPT may provide scientific rationale for the clinical use of LPT to treat inflammation and edema. Future studies will focus on the mechanism(s) responsible for these novel findings.

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