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Background Diabetic nephropathy (DN) is a major complication of diabetes mellitus. The tobacco epidemic exacerbates kidney damage in patients with DN. Clinical reports indicate that smoking is a significant risk factor for chronic kidney disease, including DN; however, the underlying molecular mechanisms remain unclear. Method In the present study, we used a diabetic mouse model to investigate the molecular mechanisms for nicotine-exacerbated DN. Twelve-week-old female mice were injected with streptozotocin (STZ) to establish a hyperglycemic diabetic model. After four months, the control and hyperglycemic diabetic mice were further divided into four groups (control, Nicotine, diabetic, Nicotine + diabetic) by intraperitoneal injection of Nicotine or PBS. After another two months, urine and blood were collected for kidney injury assay, and renal tissues were harvested for further molecular assays using RNA-seq analysis, real-time PCR, Western blot, and immunohistochemistry. In in vitro studies, we used siRNA to suppress Grem1 expression in human podocytes and then treated them with Nicotine and high glucose to compare podocyte injury. Result Nicotine administration alone did not cause apparent kidney injury, but it significantly increased hyperglycemia-induced albuminuria, BUN, and the expression of KIM-1 and NGAL. Results from RNA-seq analysis, real-time PCR, and western blot analysis revealed that, compared to hyperglycemia or Nicotine alone, the combination of nicotine treatment and hyperglycemia significantly increased the expression of Grem1 and activated the TGF-β pathway. In vitro experiments, suppression of Grem1 expression attenuated nicotine-exacerbated podocyte injury. Conclusion Grem1 plays a vital role in the nicotine-exacerbated DN. Grem1 may be a potential therapeutic target for chronic smokers with DN.

Background Increased DAN protein (Grem1, Grem2, Grem3, Cerberus, NBL1, SOST, and USAG1) levels are often associated with severe disease-states in adult kidneys. Grem1, SOST, and USAG1 have been demonstrated to be upregulated and play a critical role in the progression of diabetic nephropathy (DN); however, the expression and the role of other DAN family members in DN have not been reported yet. In this study, we investigated the expression and the role of Grem2 in the development of renal lesions in mice with type 2 DN. Methods Fourteen-week-old BTBRob/ob (a mouse model of type 2 diabetes mellitus) and control (BTBR, wild type) mice were evaluated for renal functional and structural biomarkers. Urine was collected for protein content assay, and renal tissues were harvested for molecular analysis with real-time PCR, Western blotting, and immunohistochemistry. In vitro studies, human podocytes were transfected with Grem2 plasmid and were evaluated for apoptosis (morphologic assay and Western blotting). To evaluate the Grem2-mediated downstream signaling, the phosphorylation status of Smad2/3 and Smad1/5/8 was assessed. To establish a causal relationship, the effect of SIS3 (an inhibitor for Samd2/3) and BMP-7 (an agonist for Smad1/5/8) was evaluated on Germ2-induced podocyte apoptosis. Results BTBRob/ob mice showed elevated urinary protein levels. Renal tissues of BTBRob/ob mice showed an increased expression of Grem2; both glomerular and tubular cells displayed enhanced Grem2 expression. In vitro studies, high glucose increased Grem2 expression in cultured human podocytes, whereas, Grem2 silencing partially protected podocyte from high glucose-induced apoptosis. Overexpression of Grem2 in podocytes not only increased Bax/Bcl2 expression ratio but also promoted podocyte apoptosis; moreover, an overexpression of Grem2 increased the phosphorylation of Smad2/3 and decreased the phosphorylation of Smad1/5/8; furthermore, SIS3 and BMP-7 attenuated Grem2-induced podocyte apoptosis. Conclusions High glucose increases Grem2 expression in kidney cells. Grem2 mediates podocyte apoptosis through Smads.

Cancer pathogenesis involves the interplay of tumor- and microenvironment-derived stimuli. Here we focused on the influence of an immunomodulatory cell type, myeloid-derived suppressor cells (MDSCs), and their lineage-related subtypes on autologous T lymphocytes. Although MDSCs as a group correlated with an immunosuppressive Th repertoire and worse clinical course, MDSC subtypes (polymorphonuclear, PMN-MDSC, and monocytic, M-MDSCs) were often functionally discordant. In vivo, PMN-MDSCs existed in higher numbers, correlated with different Th-subsets, and more strongly associated with poor clinical course than M-MDSCs. In vitro, PMN-MDSCs were more efficient at blocking T-cell growth and promoted Th17 differentiation. Conversely, in vitro M-MDSCs varied in their ability to suppress T-cell proliferation, due to the action of TNFα, and promoted a more immunostimulatory Th compartment. Ibrutinib therapy impacted MDSCs differentially as well, since after initiating therapy, PMN-MDSC numbers progressively declined, whereas M-MDSC numbers were unaffected, leading to a set of less immunosuppressive Th cells. Consistent with this, clinical improvement based on decreasing CLL-cell numbers correlated with the decrease in PMN-MDSCs. Collectively, the data support a balance between PMN-MDSC and M-MDSC numbers and function influencing CLL disease course.

“Global patriotism” as part of global media ethics is imbued with the values of social justice and human rights for all. In relation to media values and principles, it encourages media practitioners to surpass the traditional journalism models that have been parochial, nationalist, propagandist, and divisive. This chapter argues in favor of peace journalism as a broader, value-based journalism model for journalists to face the issues and concerns faced in the twenty-first century.

To determine the role of the transplantation of bone marrow-derived mesenchymal stem cells (MSCs) in podocyte renewal, we studied BALB/C mice with or without adriamycin-induced acute kidney injury. MSCs were transplanted ectopically under the capsule of the left kidney or into the peritoneal cavity after the onset of kidney injury to test testing their local or systemic paracrine effects, respectively. Adriamycin produced increases in urine protein: creatinine ratios, blood urea nitrogen, and blood pressure, which improved after both renal subcapsular and intraperitoneal MSCs transplants. The histological changes of adriamycin kidney changes regressed in both kidneys and in only the ipsilateral kidney after intraperitoneal or renal subcapsular transplants indicating that the benefits of transplanted MSCs were related to the extent of paracrine factor distribution. Analysis of kidney tissues for p57-positive parietal epithelial cells (PECs) showed that MSC transplants restored adriamycin-induced decreases in the abundance of these cells to normal levels, although after renal subcapsular transplants these changes did not extend to contralateral kidneys. Moreover, adriamycin caused inflammatory activation of PECs with coexpression of CD44 and phospho-ERK, which was normalized in both or only ipsilateral kidneys depending on whether MSCs were transplanted in the peritoneal cavity or subcapsular space, respectively.

Adipose tissue is a dynamic metabolic organ that is essential for the regulation of whole-body glucose homeostasis. Adipose tissue hypertrophy with obesity is associated with glucose intolerance and insulin resistance underlying type 2 diabetes. Furthermore, studies suggest fibrosis formation is increased in adipose tissue of mice fed with a high-fat diet (HFD) and results in metabolically dysfunctional adipose tissue. Yes-associated protein 1 (YAP) is a transcription cofactor that promotes fibrosis in the lung, liver, and kidney. However, the role of YAP in adipose tissue and glucose homeostasis is unknown. To understand the role of YAP in vivo under metabolic stress conditions, we assessed how HFD impacts YAP protein levels. We have found that YAP protein levels increase in adipose tissue with weight gain. This suggests that YAP signaling may contribute to adipocyte dysfunction and insulin resistance under metabolic stress conditions. To further investigate the role of YAP in adipose tissue and glucose homeostasis in vivo, we developed adipose tissue-specific YAP knockout (adipose tissue YAP-KO) mice and placed them on both chow and HFD for 12 weeks. On an HFD, adipose tissue YAP-KO mice show improved glucose tolerance compared to controls. While blood glucose levels were lower, adipose tissue YAP-KO mice did not show improvements in insulin sensitivity. Together, these data indicate that YAP increases in adipose tissue with weight gain and disruption of YAP in adipocytes improves glucose tolerance; suggesting that adipocyte YAP plays an important role in modulating glucose homeostasis under metabolic stress conditions. Disclosure D. Han: None. R. Aslam: None. T. Ojha: None. D. Yuen: None. C.T. Luk: None.

EDA2R is a member of the large family of tumor necrosis factor receptor (TNFR). Previous studies suggested that EDA2R expression might be increased in the kidneys of diabetic mice. However, its mRNA and protein expression in kidneys were not analyzed; moreover, its role in the development of diabetic kidney disease was not explored. Here we analyzed the mRNA and protein expressions of EDA2R in diabetic kidneys and examined its role in the podocyte injury in high glucose milieu. By analysis with real-time PCR, Western blotting, we found that both the mRNA and protein levels of EDA2R were increased in the kidneys of diabetic mice. Immunohistochemical studies revealed that EDA2R expression was enhanced in both glomerular and tubular cells of diabetic mice and humans. In vitro studies, high glucose increased EDA2R expression in cultured human podocytes. Overexpression of EDA2R in podocytes promoted podocyte apoptosis and decreased nephrin expression. Moreover, ED2AR increased ROS generation in podocytes, while inhibiting ROS generation attenuates EDA2R-mediated podocyte injury. In addition, EDA2R silencing partially suppressed high glucose-induced ROS generation, apoptosis, and nephrin decrease. Our study demonstrated that high glucose increases EDA2R expression in kidney cells and that EDA2R induces podocyte apoptosis and dedifferentiation in high glucose milieu partially through enhanced ROS generation.

The loss of podocyte (PD) molecular phenotype is an important feature of diabetic podocytopathy. We hypothesized that high glucose (HG) induces dedifferentiation in differentiated podocytes (DPDs) through alterations in the apolipoprotein (APO) L1-microRNA (miR) 193a axis. HG-induced DPD dedifferentiation manifested in the form of downregulation of Wilms’ tumor 1 (WT1) and upregulation of paired box 2 (PAX2) expression. WT1-silenced DPDs displayed enhanced expression of PAX2. Immunoprecipitation of DPD cellular lysates with anti-WT1 antibody revealed formation of WT1 repressor complexes containing Polycomb group proteins, enhancer of zeste homolog 2, menin, and DNA methyltransferase (DNMT1), whereas silencing of either WT1 or DNMT1 disrupted this complex with enhanced expression of PAX2. HG-induced DPD dedifferentiation was associated with a higher expression of miR193a, whereas inhibition of miR193a prevented DPD dedifferentiation in HG milieu. HG downregulated DPD expression of APOL1. miR193a-overexpressing DPDs displayed downregulation of APOL1 and enhanced expression of dedifferentiating markers; conversely, silencing of miR193a enhanced the expression of APOL1 and preserved DPD phenotype. Moreover, stably APOL1G0-overexpressing DPDs displayed the enhanced expression of WT1 but attenuated expression of miR193a; nonetheless, silencing of APOL1 reversed these effects. Since silencing of APOL1 enhanced miR193a expression as well as dedifferentiation in DPDs, it appears that downregulation of APOL1 contributed to dedifferentiation of DPDs through enhanced miR193a expression in HG milieu. Vitamin D receptor agonist downregulated miR193a, upregulated APOL1 expression, and prevented dedifferentiation of DPDs in HG milieu. These findings suggest that modulation of the APOL1-miR193a axis carries a potential to preserve DPD molecular phenotype in HG milieu.

Human parietal epithelial cells (PECs) are progenitor cells that sustain podocyte homeostasis. We hypothesized that the lack of apolipoprotein (APO) L1 ensures the PEC phenotype, but its induction initiates PEC transition (expression of podocyte markers). APOL1 expression and down-regulation of miR193a coincided with the expression of podocyte markers during the transition. The induction of APOL1 also stimulated transition markers in human embryonic kidney cells (cells with undetectable APOL1 protein expression). APOL1 silencing in PECs up-regulated miR193a expression, suggesting the possibility of a reciprocal feedback relationship between APOL1 and miR193a. HIV, interferon-γ, and vitamin D receptor agonist down-regulated miR193a expression and induced APOL1 expression along with transition markers in PECs. Luciferase assay suggested a putative interaction between miR193a and APOL1. Since silencing of APOL1 attenuated HIV-, vitamin D receptor agonist–, miR193a inhibitor–, and interferon-γ–induced expression of transition markers, APOL1 appears to be a critical functional constituent of the miR193a- APOL1 axis in PECs. This notion was confirmed by further enhanced expression of PEC markers in APOL1 mRNA–silenced PECs. In vivo studies, glomeruli in patients with HIV, and HIV/APOL1 transgenic mice had foci of PECs expressing synaptopodin, a transition marker. APOL1 likely regulates PEC molecular phenotype through modulation of miR193a expression, and APOL1 and miR193a share a reciprocal feedback relationship.

Diabetic nephropathy (DN) is a major complication of diabetes mellitus. Clinic reports indicate cigarette smoking is an independent risk factor for chronic kidney disease including DN; however, the underlying molecular mechanisms are not clear. Recent studies have demonstrated that nicotine, one of the active compounds in cigarette smoke, contributes to the pathogenesis of the cigarette smoking-accelerated chronic kidney disease. One of the characteristics of DN is the expansion of mesangium, a precursor of glomerular sclerosis. In the present study, we examined the involvement of Wnt/β-catenin pathway in nicotine-mediated mesangial cell growth in high glucose milieu. Primary human renal mesangial cells were treated with nicotine in the presence of normal (5 mM) or high glucose (30 mM) followed by evaluation for cell growth. In the presence of normal glucose, nicotine increased both the total cell numbers and Ki-67 positive cell ratio, indicating that nicotine stimulated mesangial cell proliferation. Although high glucose itself also stimulated mesangial cell proliferation, nicotine further enhanced the mitogenic effect of high glucose. Similarly, nicotine increased the expression of Wnts, β-catenin, and fibronectin in normal glucose medium, but further increased mesangial cell expression of these proteins in high glucose milieu. Pharmacological inhibition or genetic knockdown of β-catenin activity or expression with specific inhibitor FH535 or siRNA significantly impaired the nicotine/glucose-stimulated cell proliferation and fibronectin production. We conclude that nicotine may enhance renal mesangial cell proliferation and fibronectin production under high glucose milieus partly through activating Wnt/β-catenin pathway. Our study provides insight into molecular mechanisms involved in DN.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following blood transfusion. Donor antibodies are frequently involved; however, the pathogenesis and protective mechanisms in the recipient are poorly understood, and specific therapies are lacking. Using newly developed murine TRALI models based on injection of anti-major histocompatibility complex class I antibodies, we found CD4+CD25+FoxP3+ T regulatory cells (Tregs) and CD11c+ dendritic cells (DCs) to be critical effectors that protect against TRALI. Treg or DC depletion in vivo resulted in aggravated antibody-mediated acute lung injury within 90 minutes with 60% mortality upon DC depletion. In addition, resistance to antibody-mediated TRALI was associated with increased interleukin-10 (IL-10) levels, and IL-10 levels were found to be decreased in mice suffering from TRALI. Importantly, IL-10 injection completely prevented and rescued the development of TRALI in mice and may prove to be a promising new therapeutic approach for alleviating lung injury in this serious complication of transfusion.

For many years, splenectomy has been used to treat immune thrombocytopenia (ITP), and this procedure benefits approximately two-thirds of the treated patients. Although splenectomy may raise platelet counts, antibody-coated platelets and cytotoxic T lymphocytes appear to persist or can change over time. To better understand how the spleen may affect anti-platelet immune responses, we used a murine model of ITP demonstrating both antibody-mediated and T lymphocyte-mediated thrombocytopenia. Mice with severe combined immunodeficiency (SCID) were either splenectomized or not and transfused with splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 + platelets. Platelet counts and anti-platelet antibody levels were performed weekly. After 4 weeks, the mice were sacrificed, and lymphoid organs were harvested and examined by flow cytometry to quantify CD4 + CD25 + FoxP3 + Tregs and conventional cross-presenting XCR1 + and tolerizing SIRPα+ dendritic cells. The results indicate that compared with control non-splenectomized mice, thrombocytopenia was improved and anti-platelet antibody production was significantly diminished in all splenectomized mice that received immune splenocytes. Splenectomized SCID mice also had a marked reduction in Tregs in the thymus together with an increased proportion of both thymic dendritic cell subsets that correlated with increased platelet counts. Of interest, although splenectomy diminished anti-platelet antibody production and raised platelet counts, marrow megakaryocyte densities were still significantly reduced in mice that received immune splenocytes. These results suggest that the spleen in murine ITP not only is the primary site responsible for platelet destruction, but it also controls, to a significant extent, the antibody response against platelets and the migration patterns of lymphocyte subsets.

Chronic lymphocytic leukemia (CLL) is the most common form of adult leukemia in Western countries. Despite significant clinical and molecular advances, CLL remains an incurable disease. In CLL there is ongoing communication between leukemic B cells and non-leukemic cells in the tissue microenvironment. Idelalisib, a PI3Kδ inhibitor, is efficacious in CLL although side effects like inflammatory reactions in different tissues (e.g., colitis, transaminitis and pneumonitis) and progression of disease can lead to discontinuation of the drug. We previously presented an intriguing finding that a cohort of patients who discontinued idelalisib therapy due to colitis had extended survival compared to other patients who discontinued drug but did not have colitis. Of note, the colon tissues from patients with ulcerative colitis (UC) often contain elevated levels of Th17 cells and decreased levels of Treg (Foxp3+) cells. Moreover, Th17 cells from humans and mice have been shown to promote autoimmune B-cell maturation. In this regard, we have previously published that circulating Th17 levels are elevated in CLL patients and Th17 numbers correlate with extended survival. For these reasons, we have now quantified the numbers of Th17 and Treg cells in the blood and colon tissues of a cohort of CLL patients with drug-associated colitis and extended survival and have investigated the effects of in vitro exposure to idelalisib on Th17 generation. Circulating Th17A levels were quantified in a cohort of patients (n=11) before starting idelalisib-treatment and at the time the drug was discontinued due to colitis by flow cytometric analysis of intracellular IL-17A in CD4+ T cells. In addition, colon tissues from those CLL patients who developed colitis during idelalisib treatment taken at the time of drug discontinuation and patients with UC were examined to quantify the infiltration of T (CD3+) cells, Th17 (IL17A+) cells and Treg (Foxp3+) cells by immunohistochemistry (IHC). As negative control tissues (CT) for these studies we used colectomy samples from patients with inactive diverticulosis. FACS analysis of peripheral blood mononuclear cells from the CLL patients treated with idelalisib and having extended survival showed significantly higher circulating Th17A levels (P < 0.01) at the time of drug discontinuation compared to pre-treatment levels. In addition, IHC analyses on tissues from a subset of these same patients indicated that the ratio of % IL-17A+ to CD3+ cells was significantly higher in CLL than in CT (P = 0.0002). It was also significantly higher in CLL than UC (P = 0.001), even though the average number of CD3+ cells in UC was higher than in CLL (P = 0.0001). We also determined the ratio of % FoxP3+ to CD3+ cells in the colon tissues of 12 CT, 16 UC and 6 CLL patients from the cohort studied above. This revealed the average percentage of FoxP3+ cells within the total number of infiltrating T cells was significantly higher in CLL than CT (P = To examine the effect of idelalisib on Th17-cell generation, naïve CD4+ CLL T cells from 6 patients (3 M-CLL + 3 U-CLL) were activated in vitro by anti-CD3/28 ligation plus IL-2 in the presence or absence of idelalisib. After 7 days, T helper subset profiling was performed by intracellular cytokine staining. Significantly higher percentages of Th17A cells and significantly lower percentages of Th1 cells (IFNg+) (P= 0.006) were found in cultures containing idelalisib. The percentages of Tregs were unchanged in the same cultures. These findings suggest that the higher numbers of Th17s in blood and tissue of patients after idelalisib treatment may reflect the action of idelalisib on Th17 generation. In conclusion, CLL patients who discontinued idelalisib due to colitis and yet had extended survival have increased circulating and tissue-resident Th17 cells and decreased levels of Treg cells. These findings suggest that an immune imbalance between Th17 and Treg cell numbers and function promotes colitis and at the same time favors improved clinical outcome. This is consistent with the known action of Th17 cells enhancing autoimmune B-cell responses. Disclosures Barrientos: Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics/AbbVie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rai:Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Cellectis: Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Membership on an entity's Board of Directors or advisory committees. Chiorazzi:Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

T-helper 17 (Th17) cells constitute a subset of T cells that characteristically secrete IL-17. In addition to their normal adaptive immune functions, Th17 cells also play roles in supporting dysfunctional immune responses found in autoimmunity and cancer. Several studies suggest that Th17 cells play a protective role in chronic lymphocytic leukemia (CLL). For example, CLL patients exhibit varied levels of circulating Th17 cells, and elevated levels positively correlate with better clinical outcome regardless of IGHV-mutation status. To understand this relationship and elucidate the cellular and molecular mechanisms of Th17 generation in CLL, in particular the role of microRNAs known to affect Th17 development, we investigated cross-talk between naïve CD4+ T cells and CLL B cells. Moreover, since intraclonal leukemic B-cell subpopulations differing in time since cell birth/division can exhibit different functional effects on antigen presentation, we explored the effect of B-cell activation on this T - leukemic B-cell dialogue and how it affects the generation of Th17 cells. To determine potential candidates differentially expressed in CLL, we conducted genome-wide single-cell expression analysis comparing fluorescence activated cell sorting (FACS)-purified mature Th17 cells (CD3+/CD4+/CD45+/CD161+/CCR6+/ CCR4+/CXCR3-) from CLL patients and healthy donors. Selected candidate genes met the criteria of >7-fold increase in expression in CLL, adjusted p-value To determine whether CLL cells directly influence miR-155 levels in naïve CD4+ T cells, co-culture experiments using autologous leukemic or healthy B cells were carried out. FACS-purified peripheral blood naïve CD4+ T cells and B cells from CLL patients and from age-matched healthy controls were co-cultured for 3 days, and expression of T-cell miR-155 was determined by RT-qPCR. In the presence of unstimulated CLL or healthy B cells, miR-155 expression in naïve T cells remained unchanged across all co-cultures. However, upon activation, healthy and leukemic B cells exerted differential effects on miR-155 expression in autologous naïve T cells. In the presence of autologous healthy B cells pre-activated with CpG-ODN2006 and IL-15, miR-155 expression in healthy naïve T cells was significantly increased (P = 0.0313) across all samples. Conversely, CLL naïve T cells co-cultured with autologous, pre-activated leukemic B cells showed heterogeneous modulation of miR-155. Of interest, the magnitude and direction of miR-155 changes in the autologous CLL co-cultures positively correlated not only with circulating Th17 levels (P = 0.019), as determined by flow cytometry, but also with patient time to first treatment (P = 0.0003). Moreover, when samples were divided into 2 groups based on an increase or decrease in miR-155 levels after exposure to activated compared to resting CLL B cells, a significant difference was seen with median survival of 237 months and 67 months, respectively (P = 0.005). Consistent with previous observations from our lab, this correlation was independent of various prognostic markers, including IGHV-mutation status. Our results suggest the existence of a miR-155 modulatory mechanism mediated by CLL B cells that differs based on leukemic B-cell activation state and the degree of change occurring when naïve T cells are exposed to resting vs. activated B cells. Moreover, this variable effect in CLL patients differs from that in normal individuals, and the effect influences number of Th17 cells and patient outcome. Studies are underway to determine the effects that leukemic B cells, unstimulated or CpG-ODN2006 + IL-15 stimulated, have on autologous naïve T-cell maturation into Th17 cells, and the extent that this process depends on the variable miR-155 modulatory capacity of leukemic B cells. Disclosures Chiorazzi: Janssen, Inc: Consultancy; AR Pharma: Equity Ownership.

Transfusion-related acute lung injury (TRALI) is a serious complication of transfusion and has been ranked as one of the leading causes of transfusion-related fatalities. Nonetheless, many details of the immunopathogenesis of TRALI, particularly with respect to recipient factors are unknown. We used a murine model of antibody-mediated TRALI in an attempt to understand the role that recipient lymphocytes might play in TRALI reactions. Intravenous injection of an IgG2a antimurine major histocompatibility complex class I antibody (34-1-2s) into BALB/c mice induced moderate hypothermia and pulmonary granulocyte accumulation but no pulmonary edema nor mortality. In contrast, 34-1-2s injections into mice with severe combined immunodeficiency caused severe hypothermia, severe pulmonary edema, and approximately 40% mortality indicating a critical role for T and B lymphocytes in suppressing TRALI reactions. Adoptive transfer of purified CD8+ T lymphocytes or CD4+ T cells but not CD19+ B cells into the severe combined immunodeficiency mice alleviated the antibody-induced hypothermia, lung damage, and mortality, suggesting that T lymphocytes were responsible for the protective effect. Taken together, these results suggest that recipient T lymphocytes play a significant role in suppressing antibody-mediated TRALI reactions. They identify a potentially new recipient mechanism that controls the severity of TRALI reactions.

Transfusion-related immunomodulation (TRIM) has been correlated with the presence of white cells (WBCs) in blood transfusions, but the role of components such as platelets (PLTs) in mediating TRIM has not been extensively examined. We designed a murine PLT transfusion model to study whether leukoreduced PLTs mediate TRIM effects.CBA recipient mice were administered four weekly transfusions of either fresh (4 hr) or aged (24 and 72 hr) donor leukoreduced PLTs from allogeneic BALB/c mice and then transplanted with skin grafts from donor-matched mice. TRIM was measured by comparing the times to graft rejection and these were correlated with immunoglobulin G (IgG) antibody development measured by flow cytometry.Compared with nontransfused control recipients, four transfusions of fresh, extremely leukoreduced (0.05 WBCs/mL), allogeneic PLTs significantly (p0.002) reduced the recipient's ability to reject donor-matched skin grafts (survival49 days compared with14 days in nontransfused controls) despite the presence of high-titered serum IgG donor antibodies. In contrast, however, aged PLTs or fresh PLTs devoid of MHC Class I molecules were unable to affect skin graft survival nor stimulate antibody production. The PLT age-related inability to induce TRIM was shown to be due to loss of PLT-associated MHC Class I molecules; soluble supernatant MHC molecules that were transfused were unable to induce TRIM.These results suggest that fresh PLTs can induce TRIM independently of WBCs due to their MHC antigen expression whereas aging results in loss of MHC and ability to mediate TRIM. The findings support the concept that either active MHC removal from fresh PLTs or passive removal by, for example, storage, may reduce any deleterious effects of TRIM in transfusion recipients.

BACKGROUND: Anti-D treatment is effective in increasing platelet (PLT) counts in patients with autoimmune thrombocytopenic purpura (AITP); however, the exact mechanism of action is unknown. Previous results have suggested that anti-D-coated red blood cells (RBCs) affect reticuloendothelial system phagocytosis by stimulating agents (e.g., reactive oxygen species) that alter signaling pathways within the phagocyte. To address this, a flow cytometric assay was used to compare the kinetics and signaling pathways responsible for opsonized PLT and RBC phagocytosis. STUDY DESIGN AND METHODS: Human RBCs or PLTs were labeled with the fluorescent dye CM-Green, opsonized with Rh immune globulin or anti-MHC, respectively, and incubated with THP-1 monocytes with or without signal transduction inhibitors and intracellular fluorescence was analyzed. RESULTS: Compared with opsonized PLTs, phagocytosis of opsonized RBCs was significantly slower (p < 0.0001) and, within 2 hours, induced a state of phagocytic refractoriness; resting the mononuclear cells (MNCs) for up to 24 hours did not rescue their ability to further mediate PLT phagocytosis. Inhibitors of phosphatidylinositol 3-kinase (wortmannin, LY294002, myricetin, and quercetin), protein kinase C (staurosporine), and Syk kinase (piceatannol) inhibited both opsonized RBC and opsonized PLT phagocytosis. In contrast, opsonized RBC phagocytosis was significantly (p < 0.0001) enhanced by the tyrosine phosphatase inhibitor phenyl arsine oxide, whereas PLT phagocytosis was significantly reduced (p < 0.0001). Of interest, phosphatase inhibition during opsonized RBC phagocytosis induced a longer (48 hr) phagocytic refractoriness period in the MNCs. CONCLUSION: These results suggest that the early kinetics and signaling events related to phosphatase activity regulate how mononuclear phagocytes engulf opsonized RBCs and induce phagocytic refractoriness for further PLT phagocytosis.

Toll-like receptors (TLRs) play a critical role in stimulating innate immunity by recognizing pathogen-associated molecular patterns (PAMPs) on invading microorganisms. Platelets also play a role in innate immunity, and we studied whether they express TLR. Results show that human and murine platelets variably expressed TLR2, TLR4, and TLR9 by flow cytometry and Western blotting. TLR4 expression was confirmed by demonstrating murine platelet binding to lipopolysaccharide (LPS). Thrombin activation of the platelets significantly enhanced the expression of TLR9, suggesting that at least some TLRs may derive from intracellular compartments. When LPS was administered to LPS-sensitive C3H/HeN and LPS-resistant C3H/HeJ mice, functional TLR4 expression in vivo was shown to be responsible for LPS-induced thrombocytopenia. However, when the C3H/HeN mice were first rendered thrombocytopenic by an antiplatelet antibody and then administered LPS, a significant reduction occurred in their ability to produce TNF-α. The decreased cytokine production in the thrombocytopenic mice was restored with platelet transfusion. These results suggest that platelets express various TLRs and that the functional significance of one of these, TLR4, appears to be a role in the modulation of LPS-induced thrombocytopenia and TNF-α production. This work implicates platelets as important mediators of innate immune responses against invading microorganisms.

Platelets express Toll-like receptor 4 (TLR4), and this has been shown to be responsible for the thrombocytopenia induced by lipopolysaccharide (LPS) administration in vivo. We studied the role of LPS in mediating platelet phagocytosis by THP-1 cells in vitro by flow cytometry. Opsonization of platelets with an IgG monoclonal (W6/32) antibody or with IgG autoantibody-positive sera from patients with autoimmune thrombocytopenia (AITP) significantly enhanced platelet phagocytosis (P < .001). In contrast, platelet phagocytosis did not occur if platelets were bound with only LPS. If, however, the LPS-bound platelets were also opsonized with either W6/32 or autoantibody-positive sera with titers greater than 4, there was a significant and synergistic increase in Fc-dependent platelet phagocytosis (P < .001, P = .003, P = .048, and P = .047). These results suggest that, in the presence of antiplatelet antibodies, bacterial products can significantly alter platelet phagocytosis, and this may have relevance to how Gram-negative infections enhance platelet destruction in some patients with AITP.

Platelet transfusions are life-saving treatments for many patients with thrombocytopenia; however, their use is generally discouraged in the autoimmune disorder known as immune thrombocytopenia (ITP). We examined whether allogeneic platelet major histocompatibility complex (MHC) class I transfusions affected antiplatelet CD61-induced ITP. BALB/c CD61 knockout mice (CD61(-)/H-2(d)) were immunized against platelets from wild-type syngeneic BALB/c (CD61(+)/H-2(d)), allogeneic C57BL/6 (CD61(+)/H-2(b)), or C57BL/6 CD61 KO (CD61(-)/H-2(b)) mice, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP. When nondepleted splenocytes were transferred to induce antibody-mediated ITP, both CD61(+) platelet immunizations generated immunity that caused thrombocytopenia independently of allogeneic MHC molecules. In contrast, when B-cell-depleted splenocytes were transferred to induce T-cell-mediated ITP, transfer of allogeneic MHC-immunized splenocytes completely prevented CD61-induced ITP development. In addition, allogeneic platelet transfusions into SCID mice with established CD61-induced ITP rescued the thrombocytopenia. Compared with thrombocytopenic mice, bone marrow histology in the rescued mice showed normalized megakaryocyte morphology, and in vitro CD61-specific T-cell cytotoxicity was significantly suppressed. These results indicate that antibody-mediated ITP is resistant to allogeneic platelet transfusions, while the T-cell-mediated form of the disease is susceptible, suggesting that transfusion therapy may be beneficial in antibody-negative ITP.

TLR9 localizes to a novel intracellular compartment called the T granule to promote immune signaling by platelets., Human and murine platelets (PLTs) variably express toll-like receptors (TLRs), which link the innate and adaptive immune responses during infectious inflammation and atherosclerotic vascular disease. In this paper, we show that the TLR9 transcript is specifically up-regulated during pro-PLT production and is distributed to a novel electron-dense tubular system-related compartment we have named the T granule. TLR9 colocalizes with protein disulfide isomerase and is associated with either VAMP 7 or VAMP 8, which regulates its distribution in PLTs on contact activation (spreading). Preincubation of PLTs with type IV collagen specifically increased TLR9 and CD62P surface expression and augmented oligodeoxynucleotide (ODN) sequestration and PLT clumping upon addition of bacterial/viral ODNs. Collectively, this paper (a) tracks TLR9 to a new intracellular compartment in PLTs and (b) describes a novel mechanism of TLR9 organization and signaling in human PLTs.

Immune thrombocytopenia (ITP) is a bleeding disorder in which antibodies and/or T cells lead to enhanced peripheral platelet destruction and reduced bone marrow platelet production. Several reports have observed that ITP is associated with a peripheral deficiency of tolerance-inducing CD4+CD25+FoxP3+ T regulatory cells (Tregs). Using a murine model of ITP, we analyzed Tregs in the spleen and thymus. CD61 knockout mice were immunized against wild-type (CD61+) platelets, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice. Compared with SCID mice receiving naive splenocytes, within 2 weeks after transfer, the ITP SCID mice became thrombocytopenic (< 200 × 109 platelets/L) and had increased serum anti-CD61 antibodies. The quantity of thymic Tregs by 2 weeks after transfer was significantly elevated, whereas Tregs in the spleens were significantly reduced. Treatment of the ITP mice with 2 g/kg intravenous immunoglobulin raised the platelet counts, reduced antibody production, and normalized the thymic and splenic Treg populations. Compared with thymocytes from ITP mice treated with intravenous immunoglobulin, thymocytes from untreated ITP mice delayed the onset of ITP when administered before engraftment with immune splenocytes. These results suggest that ITP in mice is associated with a peripheral Treg deficiency because of thymic retention and therapy normalizes the Tregs.

Immune thrombocytopenia (ITP) is a bleeding disorder characterized by antibody-opsonized platelets being prematurely destroyed in the spleen, although some patients with ITP may have a cell-mediated form of thrombocytopenia. Although several animal models of ITP have been developed, few mimic primary chronic ITP nor have any shown cell-mediated platelet destruction. To create this type of model, splenocytes from CD61 knockout mice immunized against CD61+ platelets were transferred into severe combined immunodeficient (SCID) (CD61+) mouse recipients, and their platelet counts and phenotypes were observed. As few as 5 × 104 splenocytes induced a significant thrombocytopenia and bleeding mortality (80%) in recipients within 3 weeks after transfer. Depletion of lymphocyte subsets before transfer showed that the splenocyte's ability to induce thrombocytopenia and bleeding completely depended on CD4+ T helper cells and that both CD19+ B cell (antibody)– and CD8+ T cell (cell)–mediated effector mechanisms were responsible. Treatment of the SCID mouse recipients with intravenous γ-globulins raised platelet counts and completely prevented bleeding mortality induced by antibody-mediated effector mechanisms but did not affect cell-mediated disease. This novel model not only shows both antibody- and cell-mediated ITP and bleeding but also suggests that these 2 effector mechanisms have a differential response to therapy.

1. Human plasma contains unidentified components that inhibit arachidonic acid (AA) metabolism. In the present study, we investigated whether plasma from rabbits fed a normal or high-cholesterol diet for 16 weeks also inhibits AA metabolism. Specifically, we studied the effects of plasma on platelet aggregation and on the production of AA metabolites, tri-hydroxyeicosatrienoic acid, 12-hydroxyeicosatetraenoic acid and thromboxane B(2). 2. Haematological and lipid profiles were altered by a high-cholesterol diet. Platelets from hypercholesterolaemic rabbits showed enhanced aggregatory sensitivity to AA and platelet-activating factor. However, plasma from hypercholesterolaemic and control rabbits, when added to the incubation mixture, significantly inhibited platelet aggregation and eicosanoid production. 3. High- and low-density lipoprotein (HDL and LDL, respectively) concentrations increased several-fold in plasma with cholesterol feeding. When added directly to the incubation mixture, both HDL and LDL inhibited platelet aggregation, as well as AA metabolism. 4. Haptoglobin, albumin and Cohn's fraction IV, but not globulins, exhibited antiplatelet and anti-AA metabolism activities. Their concentrations in plasma were not affected by cholesterol feeding. 5. We conclude that LDL and HDL account for at least some of the inhibition of AA metabolism produced by plasma.

Rh immune globulin (WinRho SDF; Cangene, Mississauga, ON, Canada) is an effective treatment for autoimmune thrombocytopenic purpura; however, maintaining a sustained supply for its use in autoimmune thrombocytopenic purpura and its primary indication, hemolytic disease of the newborn, makes the development of alternative reagents desirable. We compared Rh immune globulin and 6 human monoclonal anti-D antibodies (MoAnti-D) with differing isotypes and specificities for their ability to opsonize erythrocytes and inhibit platelet phagocytosis in an in vitro assay. Results demonstrated that opsonization of erythrocytes with Rh immune globulin significantly (P < .001) reduced phagocytosis of fluorescently labeled opsonized platelets in an Fc-dependent manner. Of the MoAnti-D that shared specificity but differed in isotype, only IgG3 antibodies could significantly (P < .001) inhibit platelet phagocytosis. In contrast, 2 MoAnti-D shared isotypes and differed in specificity; however, only one could significantly (P < .001) inhibit platelet phagocytosis. The results suggest that MoAnti-D epitope specificity and isotypes are critical requirements for optimal inhibition of opsonized platelet phagocytosis.

Transfusion-related acute lung injury (TRALI), a syndrome characterized by respiratory distress triggered by blood transfusions, is the leading cause of transfusion-related mortality. Mostly, TRALI has been attributed to passive infusion of human leucocyte antigen (HLA) and human neutrophil antigen (HNA) antibodies present in the transfused blood product. Several animal models have been developed to study the pathogenesis of antibody-mediated TRALI and various mechanisms for TRALI induction have been suggested, including involvement of endothelial cells, neutrophils and monocytes. In 2006, a murine of model of antibody-mediated TRALI was developed using a monoclonal MHC class I antibody (clone 34-1-2s). This antibody was shown to cause significant lung damage (excess lung water: pulmonary edema) within 2 hours of administration into BALB/c mice, which in follow-up studies was only reproducible after initial priming with the gram-negative endotoxin lipopolysaccharide (LPS). 34-1-2s was also shown to cause severe lung damage in severe combined immunodeficient (SCID) mice. We investigated 34-1-2s mediated TRALI in BALB/c mice, without LPS priming, and found no difference in TRALI severity when compared with injection with an control isotype antibody for 34-1-2s (Isotype Mouse IgG2a antibody), as examined by lung wet-to-dry ratios, a measure for pulmonary edema. Recently it was described that the acute phase protein C-reactive protein (CRP), heavily up-regulated during acute infections and also present at lower levels in healthy individuals, was able to enhance antibody-mediated platelet destruction both in vitro and in vivo via Fc-receptor mediated phagocytic responses. Considering the fact that TRALI has been shown to be mainly antibody-mediated, plus the fact that it has been suggested to be an Fc-dependent process as well, we investigated the effect of CRP in a murine antibody-mediated TRALI. We tested if CRP would be able to enhance antibody-mediated TRALI in the murine 34-1-2s based BALB/c TRALI model. For that purpose, we co-injected CRP together with 34-1-2s and compared that to co-injection of CRP together with control isotype mouse IgG2a or to injection with CRP alone. We found that CRP+34-1-2s injection resulted in significantly higher lung damage than CRP+isotype antibody, as well as than CRP alone, with at least 43% of the mice in the CRP+34-1-2s group having a lung wet-to-dry ratio of higher than 5, which is considered to represent severe lung damage. As the monocyte-derived neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2: murine equivalent of human IL-8) was recently shown to play a central role in murine (SCID) 34-1-2s-mediated TRALI induction, we measured MIP-2 values in our BALB/c TRALI model and found that CRP alone was capable of producing high levels of MIP-2, which were found to be even more increased when 34-1-2s was co-injected with CRP. We propose a mechanism in which CRP plays a synergistic role with 34-1-2s antibody to significantly increase the induction of antibody-mediated TRALI via enhanced stimulation of monocyte-derived MIP-2 secretion. Disclosures No relevant conflicts of interest to declare.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder in which autoantibodies and autoreactive T cells target the destruction of platelets and megakaryocytes in the spleen and bone marrow. Several therapeutic options e.g. steroids, IVIg, Rituximab and thrombopoietin mimetics, are available for patients but inadequate efficacy, side effects and/or expense can make them undesirable. PRTX-100 is a highly purified formulation of Staphylococcal Protein A that has been evaluated in clinical trials with rheumatoid arthritis patients. Here, we analyzed the efficacy of PRTX-100 in raising platelet counts in a well-established murine model of ITP that demonstrates both antibody- and T cell-mediated thrombocytopenia. Platelet glycoprotein (GP) IIIa (CD61) knockout (KO) mice were immunized with CD61+ platelets and ITP was initiated by transfer of their splenocytes into severe combined immunodeficient (SCID) mice. On day 10, post transfer, the SCID mice were treated with either placebo, 1g/kg IVIg (ip, biweekly) or doses of PRTX-100 (iv, biweekly, 2.5-250 ug/kg) and platelet counts were measured weekly. Results show that control SCID mice transferred with 1x104 splenocytes from immune CD61 KO mice became thrombocytopenic at day 7 post-transfer and remained extremely thrombocytopenic throughout the 28 day protocol (3 mice died from bleeding diatheses). Similar observations were found with SCID mice given placebo. In contrast, however, transferred SCID mice treated with either IVIg or the PRTX-100 doses had platelet counts that increased to within normal levels within 1-2 weeks after treatment and none of the mice died (Table 1). Table 1.Platelet counts (mean+SD) in SCID ITP mice (N=3-8/group) treated with the indicated compounds.TreatmentPre-bleedDay 14Day 21Day 28No treatment848+20289+4651+4278+51Placebo (250 ug/kg)848+202113+5946+2063+40IVIg (1 g/kg)848+202290+211490+289698+350PRTX-100 (250 ug.kg)848+202101+37303+112505+226PRTX-100 (25 ug.kg)848+202203+88290+67254+70PRTX-100 (2.5 ug.kg)848+202275+202405+289631+401 These results demonstrate that PRTX-100 was effective in elevating platelet counts in a murine model of human ITP and support the proof of principle that PRTX-100 may be beneficial in patients with ITP. Disclosures No relevant conflicts of interest to declare.

Immune thrombocytopenia (ITP) is an autoimmune bleeding disorder characterized by low platelet counts. ITP has a complex pathogenesis, in which both anti-platelet antibodies as well as T cells have been shown to be important. Initial management of newly diagnosed ITP may be either watchful waiting or pharmacologic intervention, such as glucocorticoids or Intravenous Immunoglobulin (IVIg), a blood product consisting of polyclonal immunoglobulin G (IgG) derived from thousands of donors. Second-line therapy may include dexamethasone, high-dose methylprednisolone, rituximab, thrombopoietin (TPO)-receptor agonists, or splenectomy. The working mechanism of IVIg is actively under investigation and is still a matter of debate, as various different working mechanisms have been suggested. One of them is that IVIg may shift the balance from a pro- to anti-inflammatory state through immunomodulating the activity of dendritic cells (DCs). To gain more insights into the role of DCs in ITP, upon IVIg treatment or splenectomy, we analyzed DC subsets in a murine model of ITP, which features both the antibody and T cell mediated thrombocytopenia. Severe combined immunodeficient (SCID) mice were administrated 4x104 splenocytes from CD61 (GPIIIa) knockout mice immunized against CD61 (or naïve control splenocytes) and the mice were treated with or without 1 g/kg IVIg twice a week. Also the same type of splenocytes were transferred into splenectomized SCID mice. Weekly platelet counts were assessed and after 4 weeks the mice were sacrificed and spleen and thymuses were harvested. Splenocytes and thymocytes were isolated and examined by flow cytometry for cross-presenting (XCR1+) and non-presenting tolerizing (SIRP alpha+) DCs. Without IVIg or splenectomy, both splenic DC subset numbers correlated positively with platelet counts and both the thymic DC subset numbers correlated negatively with platelet counts, indicating thymic retention of DC in a setting of thrombocytopenia. Interestingly, splenectomized SCID mice, apart from increased platelet counts, demonstrated a complete reversal of the DC pattern in the thymus, as thymic DC subsets correlated positively with platelet counts in splenectomized mice. Upon IVIg treatment, apart from a general increase in platelet counts, the splenic tolerizing DCs significantly increased in numbers. Moreover, the thymic retention of tolerizing DCs and thus the negative correlation with platelet counts (R2: 0.46, p Disclosures No relevant conflicts of interest to declare.

Major Histocompatibility complex (MHC) class I is a complex composed of two proteins, the MHC-encoded alpha heavy chain and the beta2-microglobulin light chain. In the endoplasmic reticulum, it receives an antigen peptide of usually eight to eleven residues in its groove and is exported to the plasma membrane. In classical MHC class I presentation, endogenous antigens are presented to CD8+ T cells. On the other hand, cross-presentation consists in the presentation of exogenously-derived proteins in the context of MHC class I molecules to CD8+ T lymphocytes, inducing a cytotoxic response against the antigen presenting cell (APC). Platelets have been previously shown to be able to cross-present immunogenic ovalbumin (OVA) peptides, as well as bacterial peptides and to trigger CD8+ T lymphocyte activation. It is thought that the antigen cross presenting function of platelets is transmitted from megakaryocytes (MK) during thrombopoiesis, but whether megakaryocytes can cross-present is still unknown. To address this question, we developed an in vitro endocytosis model of murine bone marrow-derived megakaryocytes. C57BL/6 mouse megakaryocytes are enriched by gradient sedimentation and pulsed with fluorescent ovalbumin (OVA) for various times. In addition, we used DQ-OVA, a BODIPY FL-conjugated OVA, which becomes fluorescent upon proteolysis. Cells were then analyzed by flow cytometry or confocal microscopy to assess the OVA megakaryocyte internalization, its proteolytic degradation and its loading onto MHC class I molecules. We can show the endocytosis of exogenous OVA by megakaryocytes, in a pathway involving lysosomes. The endocytosed OVA were subsequently proteolytically cleaved into the immunogenic OVA peptide SIINFEKL and the peptide is inserted into the antigen binding groove of MHC class I molecules. The MHC class I-OVA peptide complexes are stored in the MK alpha-granules and can be translocated to the plasma membrane. Thus, we have shown that MKs can effectively process and present exogenous antigens on MHC class I molecules. This opens a new avenue toward a functional characterization of this pathway and its modulation, leading to new therapeutic approaches in autoimmune diseases targeting megakaryocytes, such as immune thrombocytopenia (ITP). Disclosures No relevant conflicts of interest to declare.

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related fatalities and is characterized by acute respiratory distress following transfusion of blood products. Frequently, donor antibodies present in the transfused blood product are involved, such as anti-human leukocyte antigen (HLA) antibodies or anti-human neutrophil antigen (HNA) antibodies. Several animal models of TRALI have contributed to understanding the pathogenesis which, however, is still incompletely understood. Several cell types have also been suggested to be involved in antibody-mediated TRALI, including neutrophils, endothelial cells and monocytes. Most of the animal models are based on a two-hit paradigm, where the first hit is based on "patient predisposition", such as sepsis or chronic alcohol abuse, while the second hit is delivered in the form of "transfusion factors", such as antibodies present in the transfused blood product. Although most studies have focused on factors contributing to the development of antibody-mediated TRALI, the factors and mechanisms in place to protect against antibody-mediated TRALI have been underexplored. Adoptive transfer of lymphocytes into recipient severe combined immunodeficient (SCID) mice, in which the well-established TRALI inducing anti-MHC class I antibody clone 34-1-2s was injected, was previously shown to rescue TRALI induction by 34-1-2s. Here we describe, using a murine BALB/c antibody-mediated TRALI model based on injection of 34-1-2s, that CD4 T cells, and more specifically, CD4+CD25+Foxp3+ T regulatory cells (Tregs), are responsible for protection against murine antibody-mediated TRALI. Specific in vivo depletion of CD4+ T cells, or targeted in vivo depletion of Tregs, resulted in severe lung damage after 34-1-2s infusion, as determined by increased lung wet-to-dry ratios (a measure for pulmonary edema), generally greater than 5, indicative of severe pulmonary edema. This was accompanied by significant hypothermia, increased values of the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2: equivalent of human IL-8), and increased pulmonary neutrophil accumulation, all compared to control groups. In contrast, systematic in vivo depletion of CD8+ T cells, B cells or monocytes, did not result in significant lung damage. Co-depletion of CD4+ T cells together with monocytes rescued the TRALI induction by 34-1-2s, validating the pathogenic role of monocytes in murine antibody-mediated TRALI induction. Based on MIP-2 values and in vitro studies, we suggest that Tregs suppress monocytes in order to prevent antibody-mediated TRALI. Overall, a novel first hit in TRALI induction could be identified in conditions that cause a decrease in Treg number or function, which could also explain the increased risk for human TRALI in cases of chronic alcohol abuse. In addition, therapies aimed at restoring Treg numbers or function may prove to be a novel therapeutic approach in antibody-mediated TRALI. Disclosures No relevant conflicts of interest to declare.