Gastrointestinal Flora Dictates the Biological Response in Murine Transfusion Related Acute Lung Injury (TRALI)
Authors: Kapur Rick , Kim Michael , Rebetz Johan , Takabe-French Alisa , Kim Noel , Shanmugabhavananthan Shanjeevan , Speck Edwin R. , Semple John W 
Published: April 2021

Abstract
Transfusion related acute lung injury (TRALI) is a syndrome of respiratory distress which occurs within 6 hours of blood transfusion. It is the leading cause of transfusion-related fatalities and the pathogenesis is incompletely understood. In the majority of the cases, anti-leukocyte antibodies present in the transfused blood product, in combination with recipient predisposing risk-factors such as inflammation, are implicated to be responsible for the onset of TRALI. Unfortunately, no therapies are available for TRALI. Recently, using novel murine models of TRALI, CD4+T cells were found to be important protector cells against antibody-mediated TRALI and administration of interleukin (IL)-10 was demonstrated to be a successful treatment strategy for TRALI, rescuing mice therapeutically from pulmonary edema, the hallmark of acute lung injury (Kapur et al, Blood 2017, 129(18):2557-2569). Whether the gut microbiome plays any role in the development of TRALI is currently unknown. For that purpose, we compared the biological TRALI response in mice housed in a barrier-free (BF) setting versus mice housed in a specific pathogen-free (SPF) environment. We utilized our TRALI model in which C57BL/6 mice were first depleted of CD4+ T cells in vivo followed by injection of anti-major histocompatibility complex class I antibodies (clones 34-1-2s and AF6-88.5.5.3). The TRALI response was analyzed after 90 minutes for several parameters including pulmonary edema (lung wet-to-dry weight ratios, W/Ds), rectal temperatures (indicative of systemic shock), plasma levels of macrophage inflammatory protein (MIP)-2 (murine homologue of IL-8, a neutrophil chemoattractant), levels of pulmonary neutrophils (major effector cells in TRALI) and lung histology. We observed that after one week of housing, baseline rectal temperatures of BF mice significantly increased from 38.7o C to 39.5° C (p&lt;0.001) while SPF mice remained stable around 38.3 °C. When comparing BF versus SPF mice, the baseline temperature of BF mice was significantly higher than SPF mice after one week of housing (P&lt;0.0001). The gut flora was subsequently depleted in BF mice via administration of broad-spectrum antibiotics through the drinking water. When both aerobic and anaerobic gut microbes were efficiently depleted after a week of antibiotic treatment, rectal temperatures of the treated BF mice significantly dropped again to 38.7 °C (P&lt;0.05). When inducing TRALI by depleting CD4+ T cells and injecting TRALI-antibodies, the rectal temperatures of the untreated BF mice remained low while the rectal temperatures of SPF mice and gut flora depleted-BF mice normalized again from 60 minutes onwards (34.9 versus 36.8 versus 37.8 °C, respectively). Moreover, the untreated BF mice suffered from TRALI while the SPF mice and the gut flora depleted-BF mice were protected from TRALI development (lung W/Ds 5.77 versus 4.63 versus 4.49, respectively) which was also evident from lung histology-analyses. The prevention of TRALI in SPF mice and gut flora depleted BF mice was paralleled by decreased plasma MIP-2 levels compared to the untreated BF mice which suffered from TRALI (MIP-2: 0.09 versus 0.08 versus 82.94 pg/ml, respectively) which also corresponded to pulmonary neutrophil numbers. As previously shown, low IL-10 levels were associated with TRALI development and IL-10 KO mice were found to be susceptible to antibody-mediated murine TRALI (without prior in vivo cell-depletion). However, when IL-10 KO mice were housed under SPF conditions and injected with TRALI-inducing antibodies, they did not display increased lung W/Ds levels compared to naïve SPF mice indicating that the gut flora precedes and dictates the biological TRALI response. These data collectively link the gut flora to the development of antibody-mediated TRALI in mice and antibiotic treatment prevents TRALI and may perhaps prevent the onset of human TRALI which may help in TRALI risk-assessment prior to transfusion.

Disclosures
Semple: Rigel: Consultancy; Novartis: Consultancy; Amgen: Consultancy; UCB: Consultancy.