Myeloid-derived suppressor cells prevent type 1 diabetes in murine models

Effective immunotherapy for type 1 diabetes (T1D) relies on active induction of peripheral tolerance. Myeloid-derived suppressor cells (MDSCs) play a critical role in suppressing immune responses in various pathologic settings via multiple mechanisms, including expansion of regulatory T cells (Tregs). In this study, we investigated whether MDSCs could act as APCs to induce expansion of Ag-specific Tregs, suppress T cell proliferation, and prevent autoimmune T1D development. We found that MDSC-mediated expansion of Tregs and T cell suppression required MHC-dependent Ag presentation. A murine T1D model was established in INS-HA/RAG(-/-) mice in which animals received CD4-HA-TCR transgenic T cells via adoptive transfer. We found a significant reduction in the incidence of diabetes in recipients receiving MDSC plus HA, but not OVA peptide, leading to 75% diabetes-free mice among the treated animals. To test further whether MDSCs could prevent diabetes onset in NOD mice, nondiabetic NOD/SCID mice were injected with inflammatory T cells from diabetic NOD mice. MDSCs significantly prevented diabetes onset, and 60% of MDSC-treated mice remained diabetes free. The pancreata of treated mice showed significantly lower levels of lymphocyte infiltration in islet and less insulitis compared with that of the control groups. The protective effects of MDSCs might be mediated by inducing anergy in autoreactive T cells and the development of CD4(+)CD25(+)Foxp3(+) Tregs. Thist study demonstrates a remarkable capacity of transferred MDSCs to downregulate Ag-specific autoimmune responses and prevent diabetes onset, suggesting that MDSCs possess great potential as a novel cell-based tolerogenic therapy in the control of T1D and other autoimmune diseases.     

综述:髓系衍生的抑制性细胞与肿瘤免疫耐受关系的研究进展

髓系衍生的抑制性细胞(myeloid-derived suppressor cells,MDSCs),是在肿瘤等病理因素的作用下髓系细胞发生分化障碍所产生的不同阶段髓系祖细胞的集合,具有广谱而强大的免疫抑制功能,是免疫系统的重要负性调节组件之一.研究表明:肿瘤微环境中的多种细胞因子或生长因子可通过激活相应的信号通路促进MDSCs扩增及活化,MDSCs进而通过多种机制抑制包括T细胞在内的多种免疫细胞的功能而促进肿瘤个体免疫耐受的发生.临床研究表明:肿瘤患者体内MDSCs的水平与肿瘤临床病程进展密切相关,基于MDSCs的免疫治疗也有望成为肿瘤免疫治疗的新策略.本文主要介绍了肿瘤中MDSCs的表型鉴定、扩增及活化机制、发挥免疫抑制作用的途径及机制、肿瘤中MDSCs的临床意义以及本领域需要解决的问题,以期对MDSCs在肿瘤免疫耐受中的作用进展提供参考.     

Dual roles of immune cells and their factors in cancer development and progression

Traditional wisdom holds that intact immune responses, such as immune surveillance or immunoediting, are required for preventing and inhibiting tumor development; but recent evidence has also indicated that unresolved immune responses, such as chronic inflammation, can promote the growth and progression of cancer. Within the immune system, cytotoxic CD8(+) and CD4(+) Th1 T cells, along with their characteristically produced cytokine IFN-γ, function as the major anti-tumor immune effector cells, whereas tumor associated macrophages (TAM) or myeloid-derived suppressive cells (MDSC) and their derived cytokines IL-6, TNF, IL-1β and IL-23 are generally recognized as dominant tumor-promoting forces. However, the roles played by Th17 cells, CD4(+) CD25(+) Foxp3(+) regulatory T lymphocytes and immunoregulatory cytokines such as TGF-β in tumor development and survival remain elusive. These immune cells and the cellular factors produced from them, including both immunosuppressive and inflammatory cytokines, play dual roles in promoting or discouraging cancer development, and their ultimate role in cancer progression may rely heavily on the tumor microenvironment and the events leading to initial propagation of carcinogenesis.     

Adenosinergic regulation of the expansion and immunosuppressive activity of CD11b+Gr1+ cells

Extracellular adenosine and purine nucleotides are elevated in many pathological situations associated with the expansion of CD11b(+)Gr1(+) myeloid-derived suppressor cells (MDSCs). Therefore, we tested whether adenosinergic pathways play a role in MDSC expansion and functions. We found that A(2B) adenosine receptors on hematopoietic cells play an important role in accumulation of intratumoral CD11b(+)Gr1(high) cells in a mouse Lewis lung carcinoma model in vivo and demonstrated that these receptors promote preferential expansion of the granulocytic CD11b(+)Gr1(high) subset of MDSCs in vitro. Flow cytometry analysis of MDSCs generated from mouse hematopoietic progenitor cells revealed that the CD11b(+)Gr-1(high) subset had the highest levels of CD73 (ecto-5'-nucleotidase) expression (Δmean fluorescence intensity [MFI] of 118.5 ± 16.8), followed by CD11b(+)Gr-1(int) (ΔMFI of 57.9 ± 6.8) and CD11b(+)Gr-1(-/low) (ΔMFI of 12.4 ± 1.0) subsets. Even lower levels of CD73 expression were found on Lewis lung carcinoma tumor cells (ΔMFI of 3.2 ± 0.2). The high levels of CD73 expression in granulocytic CD11b(+)Gr-1(high) cells correlated with high levels of ecto-5'-nucleotidase enzymatic activity. We further demonstrated that the ability of granulocytic MDSCs to suppress CD3/CD28-induced T cell proliferation was significantly facilitated in the presence of the ecto-5'-nucleotidase substrate 5'-AMP. We propose that generation of adenosine by CD73 expressed at high levels on granulocytic MDSCs may promote their expansion and facilitate their immunosuppressive activity.     

Pten Null Prostate Epithelium Promotes Localized Myeloid-Derived Suppressor Cell Expansion and Immune Suppression during Tumor Initiation and Progression

Chronic inflammation is known to be associated with prostate cancer development, but how epithelium-associated cancer-initiating events cross talk to inflammatory cells during prostate cancer initiation and progression is largely unknown. Using the Pten null murine prostate cancer model, we show an expansion of Gr-1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSCs) occurring intraprostatically immediately following epithelium-specific Pten deletion without expansion in hematopoietic tissues. This MDSC expansion is accompanied by sustained immune suppression. Prostatic Gr-1⁺ CD11b⁺ cells, but not those isolated from the spleen of the same tumor-bearing mice, suppress T cell proliferation and express high levels of Arginase 1 and iNOS. Mechanistically, the loss of PTEN in the epithelium leads to a significant upregulation of genes within the inflammatory response and cytokine-cytokine receptor interaction pathways, including Csf1 and Il1b, two genes known to induce MDSC expansion and immunosuppressive activities. Treatment of Pten null mice with the selective CSF-1 receptor inhibitor GW2580 decreases MDSC infiltration and relieves the associated immunosuppressive phenotype. Our study indicates that epithelium-associated tumor-initiating events trigger the secretion of inflammatory cytokines and promote localized MDSC expansion and immune suppression, thereby promoting tumor progression.     

Hepatic CD1d expression in hepatitis C virus infection and recognition by resident proinflammatory CD1d-reactive T cells

A subset of CD161(+)CD56(+/-) NKT cells can recognize glycolipids presented by CD1d and positively or negatively regulate inflammatory responses, including those implicated in several models of hepatitis. CD1d is expressed at very low levels in the healthy liver, but there is a large fraction of CD161(+)CD56(+) NKT cells. There are high levels of nonclassical proinflammatory hepatic CD1d-reactive T cells in hepatitis C virus (HCV) infection. Hepatic inflammatory cells and biliary cells adjacent to portal tract fibrotic areas of HCV-infected donors specifically up-regulated CD1d. A hepatocyte cell line expressing minimal CD1d was efficiently recognized by hepatic CD1d-reactive T cells, suggesting a role for these cells in disease. Hepatic CD1d-reactive T cells from HCV-positive as well as negative donors produced large amounts of IFN-gamma with some IL-13, but only rarely detectable IL-4. We confirmed large numbers of hepatic CD161(+) T cells, lower levels of CD56(+) T cells, and small numbers of classic invariant NKT cells. However, hepatic CD1d-reactivity was not restricted to any of these populations. We suggest virally infected hepatic cells can process potent CD1d-presented liver Ag(s), for surveillance by resident Th1 hepatic CD1d-reactive T cells. This process may be beneficial in acute viral clearance, but in chronic infection could contribute to liver injury.     

Inhibition of tumor-induced myeloid-derived suppressor cell function by a nanoparticulated adjuvant

The interaction between cancer vaccine adjuvants and myeloid-derived suppressor cells (MDSCs) is currently poorly understood. Very small size proteoliposomes (VSSP) are a nanoparticulated adjuvant under investigation in clinical trials in patients with renal carcinoma, breast cancer, prostate cancer, and cervical intraepithelial neoplasia grade III. We found that VSSP adjuvant induced a significant splenomegaly due to accumulation of CD11b(+)Gr-1(+) cells. However, VSSP-derived MDSCs showed a reduced capacity to suppress both allogeneic and Ag-specific CTL response compared with that of tumor-induced MDSCs. Moreover, splenic MDSCs isolated from tumor-bearing mice treated with VSSP were phenotypically more similar to those isolated from VSSP-treated tumor-free mice and much less suppressive than tumor-induced MDSCs, both in vitro and in vivo. Furthermore, different from dendritic cell vaccination, inoculation of VSSP-based vaccine in EG.7-OVA tumor-bearing mice was sufficient to avoid tumor-induced tolerance and stimulate an immune response against OVA Ag, similar to that observed in tumor-free mice. This effect correlated with an accelerated differentiation of MDSCs into mature APCs that was promoted by VSSP. VSSP used as a cancer vaccine adjuvant might thus improve antitumor efficacy not only by stimulating a potent immune response against tumor Ags but also by reducing tumor-induced immunosuppression.     

Circulating myeloid-derived suppressor cells: An independent prognostic factor in patients with breast cancer

Evading immune destruction is a hallmark of cancer. Myeloid-derived suppressor cells (MDSCs), a heterogeneous population of myeloid immune cells, are thought to foster the establishment of an immunosuppressive tumor microenvironment, but it remains unclear how. This study aims to determine the levels of circulating MDSCs and their subpopulations and test their immunosuppressive functions in patients with breast cancer (BC). We analyzed the fractions of MDSCs in freshly isolated peripheral blood mononuclear cells of patients with BC and healthy donors using flow cytometry. Circulating MDSCs were further phenotyped using fluorescently labeled antihuman monoclonal antibodies. Coculture experiments revealed the effects of MDSCs on CD3⁺ T cell response. Moreover, we correlated circulating MDSC levels with clinicopathological features of patients with BC. We show that the fraction of HLA-DR ^– CD33 ⁺ MDSCs in peripheral blood is about 10-fold higher in patients with BC than in healthy control individuals. The levels of all MDSC subpopulations, including monocytic and granulocytic MDSCs, are significantly elevated. Coculture experiments of purified HLA-DR ^– CD33 ⁺ MDSCs and CD3 ⁺ T cells demonstrate that T cell proliferation is more effectively inhibited by BC patient-derived MDSCs than by healthy control MDSCs. Moreover, increased circulating MDSC levels robustly associate with advanced BC stage and positive lymph node status. By being more abundant and more effective T cell suppressors, BC patient-derived circulating MDSCs exert a dual immunosuppressive effect. Our findings pave the way to develop novel diagnostic and immunotherapeutic strategies, aimed at detecting and inhibiting MDSCs in patients with BC.     

Chronic intake of high fish oil diet induces myeloid-derived suppressor cells to promote tumor growth

Omega-3 polyunsaturated fatty acids enriched fish oil exerts beneficial anti-inflammatory effects in animal models with acute and chronic inflammatory diseases. Myeloid-derived suppressor cells (MDSCs), comprised of myeloid progenitors and precursors of myeloid cells, play vital roles in cancer. How fish oil affects the generation of MDSCs and the tumor development remains largely unexplored. Here, we show that dietary intake of high fish oil diet suppresses CD8⁺ T cells activation and proliferation in vivo via elevated levels of MDSCs. Mechanistically, high fish oil diet induces the expression of immunosuppressive cytokine IL-10 and promotes myelopoiesis in the spleen as well as other peripheral tissues. The immature myeloid cells in the spleen exhibit morphological and functional characteristics of MDSCs with the capability to downregulate CD8⁺ T cells activation. Depletion of MDSCs using anti-Gr-1 antibody decreases the growth of subcutaneously transferred B16 melanoma in mice on high fish oil diet. Interestingly, diet-induced production of MDSCs is not solely dependent of the spleen, as splenectomy has no effect on the tumor progress. Our data show that the liver functions as an alternative extramedullary hematopoiesis organ to support MDSCs differentiation and maintain tumor growth. Taken together, our study provides a novel insight into the physiological effects of fish oil and points to MDSCs as a possible mediator linking dietary fish oil intake and immunosuppression in cancer immunosurveillance.     

Thymic stromal lymphopoietin is a key mediator of breast cancer progression

Inflammation is a double-edged sword that can promote or suppress cancer progression. In this study, we report that thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs, is also expressed in human and murine cancers. Our studies with murine cancer cells indicate that TSLP plays an essential role in cancer escape, as its inactivation in cancer cells alone was sufficient to almost completely abrogate cancer progression and lung metastasis. The cancer-promoting activity of TSLP primarily required signaling through the TSLP receptor on CD4(+) T cells, promoting Th2-skewed immune responses and production of immunosuppressive factors such as IL-10 and IL-13. Expression of TSLP therefore may be a useful prognostic marker, and its targeting could have therapeutic potential.     

Myeloid-derived suppressor cells and the pathogenesis of human immunodeficiency virus infection

There are several mechanisms by which human immunodeficiency virus (HIV) can mediate immune dysfunction and exhaustion during the course of infection. Chronic immune activation, after HIV infection, seems to be a key driving force of such unwanted consequences, which in turn worsens the pathological status. In such cases, the immune system is programmed to initiate responses that counteract unwanted immune activation, for example through the expansion of myeloid-derived suppressor cells (MDSCs). Although the expansion of immune suppressor cells in the setting of systemic chronic immune activation, in theory, is expected to contain immune activation, HIV infection is still associated with a remarkably high level of biomarkers of immune activation. Paradoxically, the expansion of immune suppressor cells during HIV infection can suppress potent anti-viral immune responses, which in turn contribute to viral persistence and disease progression. This indicates that HIV hijacks not only immune activation but also the immune regulatory responses to its advantage. In this work, we aim to pave the way to comprehend how such unwanted expansion of MDSCs could participate in the pathology of acute/primary and chronic HIV infection in humans, as well as simian immunodeficiency virus infection in rhesus macaques, according to the available literature.     

Both miR-17-5p and miR-20a alleviate suppressive potential of myeloid-derived suppressor cells by modulating STAT3 expression

Myeloid-derived suppressor cells (MDSCs) were one of the major components of the immune suppressive network. STAT3 has an important role in regulating the suppressive potential of MDSCs. In this study, we found that the expression of STAT3 could be modulated by both miR-17-5p and miR-20a. The transfection of miR-17-5p or miR-20a remarkably reduces the expression of reactive oxygen species and the production of H(2)O(2), which are regulated by STAT3. MDSCs transfected with miR-17-5p or miR-20a are less able to suppress Ag-specific CD4 and CD8 T cells. Importantly, both miR-17-5p and miR-20a alleviate the suppressive function of MDSCs in vivo. The expression of miR-17-5p and miR-20a in tumor-associated MDSCs was found to be lower than in Gr1(+)CD11b(+) cells isolated from the spleens of disease-free mice. Tumor-associated factor downregulates the expression of both miR-17-5p and miR-20a. The modulation of miR-17-5p and miR-20a expression may be important for the process by which patients with a tumor can overcome the immune tolerance mediated by MDSCs. Our results suggest that miR-17-5p and miR-20a could potentially be used for immunotherapy against diseases, especially cancer, by blocking STAT3 expression.     

Swertianolin ameliorates immune dysfunction in sepsis via blocking the immunosuppressive function of myeloid-derived suppressor cells

In this study, we studied the long-term proliferation trajectory of myeloid-derived suppressor cells (MDSCs) in murine sepsis model and investigated whether swertianolin could modulate the immunosuppressive function of MDSCs. A murine sepsis model was established by cecal ligation and perforation (CLP), according to the Minimum Quality Threshold in Pre-Clinical Sepsis Studies (MQTiPSS) guidelines. The bone marrow and spleen of the mice were collected at 24 h, 72 h, 7 and 15 d after sepsis induction. The proportions of monocytic- MDSCs (M-MDSCs; CD11b⁺LY6G^–LY6C^hi) and granulocytic-MDSCs (G-MDSC, CD11b⁺ Ly6G⁺ Ly6C^low) were analyzed by flow cytometry. Then, we have investigated whether swertianolin could modulate the immunosuppressive function of MDSCs in in vitro experiments. G-MDSCs and M-MDSCs increased acutely after sepsis with high levels sustained over a long period of time. G-MDSCs were the main subtype identified in the murine model of sepsis with polymicrobial peritonitis. Furthermore, it was found that swertianolin reduced significantly interleukin-10 (IL-10), nitric oxide (NO), reactive oxygen species (ROS), and arginase production in MDSCs, while reducing MDSC proliferation and promoting MDSC differentiation into dendritic cells. Swertianolin also improved T-cell activity by blocking the immunosuppressive effect of MDSCs. Both subsets of MDSCs significantly increased in the bone marrow and spleen of the mice with sepsis, with GMDSCs being the main subtype identified. Swertianolin effectively regulated the functions of MDSCs and reduced immune suppression.Key words: Sepsis, myeloid-derived suppressor cells (MDSCs), immunosuppression, swertianolin     

Myeloid-derived suppressor cells infiltrate the heart in acute Trypanosoma cruzi infection

Chagas disease, caused by the protozoan parasite Trypanosoma cruzi, affects several million people in Latin America. Myocarditis, observed in the acute and chronic phases of the disease, is characterized by a mononuclear cell inflammatory infiltrate. We previously identified a myeloid cell population in the inflammatory heart infiltrate of infected mice that expressed arginase I. In this study, we purified CD11b(+) myeloid cells from the heart and analyzed their phenotype and function. Those CD11b(+) cells were ～70% Ly6G(-)Ly6C(+) and 25% Ly6G(+)Ly6C(+). Moreover, purified CD11b(+)Ly6G(-) cells, but not Ly6G(+) cells, showed a predominant monocytic phenotype, expressed arginase I and inducible NO synthase, and suppressed anti-CD3/anti-CD28 Ab-induced T cell proliferation in vitro by an NO-dependent mechanism, activity that best defines myeloid-derived suppressor cells (MDSCs). Contrarily, CD11b(+)Ly6G(+) cells, but not CD11b(+)Ly6G(-) cells, expressed S100A8 and S100A9, proteins known to promote recruitment and differentiation of MDSCs. Together, our results suggest that inducible NO synthase/arginase I-expressing CD11b(+)Ly6G(-) myeloid cells in the hearts of T. cruzi-infected mice are MDSCs. Finally, we found plasma l-arginine depletion in the acute phase of infection that was coincident in time with the appearance of MDSCs, suggesting that in vivo arginase I could be contributing to l-arginine depletion and systemic immunosuppression. Notably, l-arginine supplementation decreased heart tissue parasite load, suggesting that sustained arginase expression through the acute infection is detrimental for the host. This is, to our knowledge, the first time that MDSCs have been found in the heart in the context of myocarditis and also in infection by T. cruzi.     

Myeloid Derived Suppressor Cells (MDSCs) Are Increased and Exert Immunosuppressive Activity Together with Polymorphonuclear Leukocytes (PMNs) in Chronic Myeloid Leukemia Patients

Tumor immune tolerance can derive from the recruitment of suppressor cell population, including myeloid derived suppressor cells (MDSCs), able to inhibit T cells activity. We identified a significantly expanded MDSCs population in chronic myeloid leukemia (CML) patients at diagnosis that decreased to normal levels after imatinib therapy. In addition, expression of arginase 1 (Arg1) that depletes microenvironment of arginine, an essential aminoacid for T cell function, resulted in an increase in patients at diagnosis. Purified CML CD11b+CD33+CD14-HLADR- cells markedly suppressed normal donor T cell proliferation in vitro. Comparing CML Gr-MDSCs to autologous polymorphonuclear leukocytes (PMNs) we observed a higher Arg1 expression and activity in PMNs, together with an inhibitory effect on T cells in vitro. Our data indicate that CML cells create an immuno-tolerant environment associated to MDSCs expansion with immunosuppressive capacity mediated by Arg1. In addition, we demonstrated for the first time also an immunosuppressive activity of CML PMNs, suggesting a strong potential immune escape mechanism created by CML cells, which control the anti-tumor reactive T cells. MDSCs should be monitored in imatinib discontinuation trials to understand their importance in relapsing patients.     

Identification of myeloid derived suppressor cells in dogs with naturally occurring cancer

Dogs with naturally occurring cancer represent an important large animal model for drug development and testing novel immunotherapies. However, poorly defined immunophenotypes of canine leukocytes have limited the study of tumor immunology in dogs. The accumulation of myeloid derived suppressor cells (MDSCs) is known to be a key mechanism of immune suppression in tumor-bearing mice and in human patients. We sought to identify MDSCs in the blood of dogs with cancer. Peripheral blood mononuclear cells (PBMCs) from dogs with advanced or early stage cancer and from age-matched healthy controls were analyzed by flow cytometry and microscopy. Suppressive function was tested in T cell proliferation and cytokine elaboration assays. Semi-quantitative RT-PCR was used to identify potential mechanisms responsible for immunosuppression. PBMCs from dogs with advanced or metastatic cancer exhibited a significantly higher percentage of CD11b(+)CD14(-)MHCII(-) cells compared to dogs diagnosed with early stage non-metastatic tumors and healthy dogs. These CD11b(+) CD14(-)MHCII(-) cells constitute a subpopulation of activated granulocytes that co-purify with PBMCs, display polymorphonuclear granulocyte morphology, and demonstrate a potent ability to suppress proliferation and IFN-γ production in T cells from normal and tumor-bearing donors. Furthermore, these cells expressed hallmark suppressive factors of human MDSC including ARG1, iNOS2, TGF-β and IL-10. In summary our data demonstrate that MDSCs accumulate in the blood of dogs with advanced cancer and can be measured using this three-marker immunophenotype, thereby enabling prospective studies that can monitor MDSC burden.     

The early kinetics of cytomegalovirus-specific CD8+ T-cell responses are not affected by antigen load or the absence of perforin or gamma interferon

Both innate and adaptive immune responses participate in the control of murine cytomegalovirus (mCMV) infection. In some mouse strains, like BALB/c, the control of infection relies on the activities of CD8(+) T cells. mCMV-specific CD8(+) T-cell responses are unusual in that, even after mCMV has been controlled in the periphery, the numbers of circulating virus-specific CD8(+) T cells remain high compared to those observed in other viral infections. To better understand the generation and maintenance of mCMV-specific CD8(+) T-cell responses, we evaluated how antigen load and effector molecules, such as perforin (Prf) and gamma interferon (IFN-gamma), influence these responses during acute infection in vivo. Viral burden affected the magnitude, but not the early kinetics, of antigen-specific CD8(+) T-cell responses. Similarly, the magnitude of virus-specific CD8(+) T-cell expansion was affected by Prf and IFN-gamma, but contraction of antigen-specific responses occurred normally in both Prf- and IFN-gamma-deficient mice. These data indicate that control of mCMV-specific CD8(+) T-cell expansion and contraction is more complex than anticipated and, despite the role of Prf or IFN-gamma in controlling viral replication, a full program of T-cell expansion and contraction can occur in their absence.     

High mobility group protein-1 inhibits phagocytosis of apoptotic neutrophils through binding to phosphatidylserine

Phagocytosis of apoptotic cells, also called efferocytosis, is an essential feature of immune responses and critical to resolution of inflammation. Impaired efferocytosis is associated with an unfavorable outcome from inflammatory diseases, including acute lung injury and pulmonary manifestations of cystic fibrosis. High mobility group protein-1 (HMGB1), a nuclear nonhistone DNA-binding protein, has recently been found to be secreted by immune cells upon stimulation with LPS and cytokines. Plasma and tissue levels of HMGB1 are elevated for prolonged periods in chronic and acute inflammatory conditions, including sepsis, rheumatoid arthritis, acute lung injury, burns, and hemorrhage. In this study, we found that HMGB1 inhibits phagocytosis of apoptotic neutrophils by macrophages in vivo and in vitro. Phosphatidylserine (PS) is directly involved in the inhibition of phagocytosis by HMGB1, as blockade of HMGB1 by PS eliminates the effects of HMGB1 on efferocytosis. Confocal and fluorescence resonance energy transfer demonstrate that HMGB1 interacts with PS on the neutrophil surface. However, HMGB1 does not inhibit PS-independent phagocytosis of viable neutrophils. Bronchoalveolar lavage fluid from Scnn(+) mice, a murine model of cystic fibrosis lung disease which contains elevated concentrations of HMGB1, inhibits neutrophil efferocytosis. Anti-HMGB1 Abs reverse the inhibitory effect of Scnn(+) bronchoalveolar lavage on efferocytosis, showing that this effect is due to HMGB1. These findings demonstrate that HMGB1 can modulate phagocytosis of apoptotic neutrophils and suggest an alternative mechanism by which HMGB1 is involved in enhancing inflammatory responses.