The effects of oxygen and medicines on T cells in hypoxic co-culture

Objectives: Many patients with massive hemorrhage, respiratory failure due to trauma admit the emergency department, and further that the experience can fall into shock, inducing to sepsis, multiple organ failure due to hyperinflammation or immunosuppression. In the these patients, the low oxygen flow with immunosuppression is believed to play a significant role. Hence, oxygen supply and medicines is essential in severe trauma patients. Therefore, this study aims to investigate the effects of oxygen and variable medicines in hypoxic condition.

Methods: T cells and macrophages were plated into trans-well plate for co-culture for 30 minutes in hypoxia. After that, the cells were stimulated with lipopolysaccharide (LPS) followed by variable medicines by normoxia or oxygen supply for 2 hrs and cells were inculated overnight under normoxic conditions. The T cell viability was measured by MTT, and the expression of interleukin-2 (IL-2), interleukin-8 (IL-8) and macrophage migration inhibitory factor (MIF) were measured by western blots using the T cells with co-culture with inflammatory maccrophages. Also, the concentration of MIF was analyzed by ELISA.

Results: The T cells viability was decreased in hypoxia with LPS stimulation, however, pentoxifylline (PTX) effectively restored cell viability regardless of oxygen state (p < 0.05). Besides, PTX in oxygen supply status restored the decreases in IL-2 expression of T cells and the increases MIF in the LPS stimulation with hypoxia (p < 0.05).

Conclusions: PTX has more effectively restored the T cells immunosuppression in hypoxia during oxygen supply, and has an immunomodulation effect by controlling hyperinflammation.

Hypoxia; Trauma; Pentoxifylline; Co-culture; Immunosuppression

[1] Nunez TC, Cotton BA. Transfusion therapy in hemorrhage shock. Current Opinion in Critical Care. 2009; 15: 536–554.

[2] Prescott HC, Osterholzer JJ, Langa KM, Angus DC, Iwashyna TJ. Late mortality after sepsis: propensity matched cohort study. British Medical Journal. 2017; 353: i2375.

[3] Boomer JS, To K, Chang KC, Takasu O, Osborne DF, Walton AH, et al. Immunosuppression in patients who die of sepsis and multiple organ failure. Journal of the American Medical Association. 2011; 306: 2594–2605.

[4] Saito M, Inoue S, Yamashita K, Kakeji Y, Fukumoto T, Kotani J. IL-15 improves aging-induced persistent T cell exhaustion in mouse models of repeated sepsis. Shock. 2020; 53: 228–235.

[5] Junger WG, Hoyt DB, Hamreus M, Liu FC, Herdon-Remelius C, Junger W, et al. Hypertonic saline activates protein tyrosine kinases and mitogen-activated protein kinase p38 in T-cells. The Journal of Trauma. 1997; 42: 437–435.

[6] Sandoval J, Escobar J, Pereda J, Sacilotto N, Rodriguez JL, Sabater L, et al. Pentoxifylline prevents loss of PP2a phosphatase activity and recruitment of histone acetyltransferases to proinflammatory genes in acute pancreatitis. Journal of Pharmacology and Experimental Therapeutics. 2009; 331: 609–617.

[7] Yoon Y, Choi S, Hong Y, Lee S, Moon S, Cho H, et al. Effect of hypertonic saline and macrophage migration inhibitory factor in restoration of T cell dysfunction. Journal of the Korean Surgical Society. 2011; 81: 229–234.

[8] Coccia MT, Waxman K, Soliman MH, Tominaga G, Pinderski L. Pentoxifylline improves survival following hemorrhagic shock. Critical Care Medicine. 1989; 17: 36–38.

[9] Rizoli SB, Kapus A, Fan J, Li YH, Marshall C, Rotstein OD. Immunomodulatory effects of hypertonic saline resuscitation on the development of lung injury inflammation following hemorrhagic shock. Journal of Immunology. 1998; 161: 6288–6296.

[10] Abrahám IM, Harkany T, Horvath KM, Luiten PG. Action of gluco-corticoids on survival of nerve cells: promoting neurodegeneration or neuroprotection? Journal of Neuroendocrinology. 2001; 13: 749–760.

[11] Konrad FM, Neudeck G, Vollmer I, Ngamsri KC, Thiel M, Reutershan J. Protective effects of pentoxifylline in pulmonary inflammation are adenosine receptor a2a dependent. FASEB Journal. 2013; 27: 3524–3535.

[12] Bloom J, Sun S, Al-Abed Y. MIF, a controversial cytokine: a review of structural features, challenges, and opportunities for drug development. Expert Opinion on Therapeutic Targets. 2016; 20: 1463–1475.

[13] Munn DH, Shafizadeh E, Attwood JT, Bondarev I, Pashine A, Mellor AL. Inhibition of T cell proliferation by macrophage tryptophan catabolism. The Journal of Experimental Medicine. 1999; 189: 1363–1372.

[14] Schorle H, Holtschke T, Hünig T, Schimpl A, Horak I. Development and function of T cells in mice rendered interleukin-2 deficient by gene targeting. Nature. 1991; 352: 621–624.

[15] Frauwirth KA, Thompson CB. Regulation of T Lymphocyte Metabolism. The Journal of Immunology. 2004; 172: 4661–4665.

[16] Cho Y, Park S, Choi S, Yoon Y, Kim J, Lee S, et al. The inflammatory response of neutrophils in an in vitro model that approximates the postcardiac arrest state. Annals of Surgical Treatment and Research. 2017; 93: 217–224.

[17] Phipps RP, Stein SH, Roper RL. A new view of prostaglandin E regulation of the immune response. Immunology Today. 1991; 12: 349–352.

[18] Yao C, Hirata T, Soontrapa K, Ma X, Takemori H, Narumiya S. Prostaglandin E_2promotes Th1 differentiation via synergistic amplifi-cation of IL-12 signaling by cAMP and PI3-kinase. Nature Communica-tions. 2013; 4: 1685.

[19] Yoon Y, Choi S, Hong Y, Lee S, Moon S, Cho H, et al. Effect of hypertonic saline and macrophage migration inhibitory factor in restoration of T cell dysfunction. Journal of the Korean Surgical Society. 2011; 81: 229–234.

[20] Wang J, Huang M, Lee P, Komanduri K, Sharma S, Chen G, et al. Interleukin-8 inhibits non-small cell lung cancer proliferation: a possible role for regulation of tumor growth by autocrine and paracrine pathways. Journal of Interferon & Cytokine Research. 1996; 16: 53–60.

[21] Rotondo R, Barisione G, Mastracci L, Grossi F, Orengo AM, Costa R, et al. IL-8 induces exocytosis of arginase 1 by neutrophil polymorphonucle-ars in nonsmall cell lung cancer. International Journal of Cancer. 2009; 125: 887–893.