BackgroundMultiple sclerosis (MS) is a chronic inflammatory autoimmune disease of the central nervous system (CNS). One potential therapeutic strategy for MS is to induce regulatory cells that mediate immunological tolerance. Probiotics, including lactobacilli, are known to induce immunomodulatory activity with promising effects in inflammatory diseases. We tested the potential of various strains of lactobacilli for suppression of experimental autoimmune encephalomyelitis (EAE), an animal model of MS.Methodology/Principal FindingsThe preventive effects of five daily-administered strains of lactobacilli were investigated in mice developing EAE. After a primary screening, three Lactobacillus strains, L. paracasei DSM 13434, L. plantarum DSM 15312 and DSM 15313 that reduced inflammation in CNS and autoreactive T cell responses were chosen. L. paracasei and L. plantarum DSM 15312 induced CD4+CD25+Foxp3+ regulatory T cells (Tregs) in mesenteric lymph nodes (MLNs) and enhanced production of serum TGF-β1, while L. plantarum DSM 15313 increased serum IL-27 levels. Further screening of the chosen strains showed that each monostrain probiotic failed to be therapeutic in diseased mice, while a mixture of the three lactobacilli strains suppressed the progression and reversed the clinical and histological signs of EAE. The suppressive activity correlated with attenuation of pro-inflammatory Th1 and Th17 cytokines followed by IL-10 induction in MLNs, spleen and blood. Additional adoptive transfer studies demonstrated that IL-10 producing CD4+CD25+ Tregs are involved in the suppressive effect induced by the lactobacilli mixture.Conclusions/SignificanceOur data provide evidence showing that the therapeutic effect of the chosen mixture of probiotic lactobacilli was associated with induction of transferable tolerogenic Tregs in MLNs, but also in the periphery and the CNS, mediated through an IL-10-dependent mechanism. Our findings indicate a therapeutic potential of oral administration of a combination of probiotics and provide a more complete understanding of the host-commensal interactions that contribute to beneficial effects in autoimmune diseases.

Acute lung injury (ALI) is an inflammatory disease with a high mortality rate. Although typically seen in individuals with sepsis, ALI is also a major complication in severe acute pancreatitis (SAP). The pathophysiology of SAP-associated ALI is poorly understood, but elevated serum levels of IL-6 is a reliable marker for disease severity. Here, we used a mouse model of acute pancreatitis-associated (AP-associated) ALI to determine the role of IL-6 in ALI lethality. Il6-deficient mice had a lower death rate compared with wild-type mice with AP, while mice injected with IL-6 were more likely to develop lethal ALI. We found that inflammation-associated NF-κB induced myeloid cell secretion of IL-6, and the effects of secreted IL-6 were mediated by complexation with soluble IL-6 receptor, a process known as trans-signaling. IL-6 trans-signaling stimulated phosphorylation of STAT3 and production of the neutrophil attractant CXCL1 in pancreatic acinar cells. Examination of human samples revealed expression of IL-6 in combination with soluble IL-6 receptor was a reliable predictor of ALI in SAP. These results demonstrate that IL-6 trans-signaling is an essential mediator of ALI in SAP across species and suggest that therapeutic inhibition of IL-6 may prevent SAP-associated ALI. IntroductionAcute pancreatitis (AP) accounts for more than 220,000 hospital admissions in the United States each year. Risk factors for AP include gallstones and excessive alcohol use. Interestingly, 70%-80% of AP patients develop mild and uncomplicated AP, while 20%-30% will develop more severe symptoms with concomitant multiple organ failure (MOF) (1). MOF is a consequence of the systemic activation of the immune system, known as systemic inflammatory response syndrome (SIRS). The clinical and pathological features of SIRS mimic those of sepsis; however, efforts to identify any infecting organisms in many patients with SIRS have failed (2-4). Although this syndrome is typically seen in individuals with sepsis, SIRS also occurs in patients with severe AP (SAP), blunt trauma, aseptic burns, and widespread surgical manipulations (5, 6). A major complication during SAP is acute lung injury (ALI). Nevertheless, the clinical course of ALI in SAP is still unpredictable and has a mortality rate of up to 50%. Current therapeutic approaches in SAP and associated ALI are symptomatically based (1, 7).The pathophysiology of SAP with ALI is poorly understood. Researchers have long hypothesized that SAP results from activation of digestive enzymes within the pancreas, a process called autodigestion (8). Indeed, inherited mutations in genes encoding for digestive enzymes have been found in patients with a hereditary form of pancreatitis. However, all these patients develop chronic pancreatitis, rather than SAP with ALI (9, 10). Therefore,

NETs form in the pancreata of mice during the development of AP, and NET levels are increased in plasma from patients with AP, compared with controls. NETs regulate organ inflammation and injury in mice with AP, and might be targeted to reduce pancreatic tissue damage and inflammation in patients.

These data demonstrate that platelets play a key role in regulating infiltration of neutrophils and edema formation in the lung via upregulation of Mac-1 in abdominal sepsis.

Neutrophil-mediated lung damage is an insidious feature in septic patients, although the adhesive mechanisms behind pulmonary recruitment of neutrophils in polymicrobial sepsis remain elusive. The aim of the present study was to define the role of lymphocyte function antigen-1 (LFA-1) and membrane-activated complex 1 (Mac-1) in septic lung injury. Pulmonary edema, bronchoalveolar infiltration of neutrophils, levels of myeloperoxidase, and CXC chemokines were determined after cecal ligation and puncture (CLP). Mice were treated with monoclonal antibodies directed against LFA-1 and Mac-1 before CLP induction. Cecal ligation and puncture induced clear-cut pulmonary damage characterized by edema formation, neutrophil infiltration, and increased levels of CXC chemokines in the lung. Notably, immunoneutralization of LFA-1 or Mac-1 decreased CLP-induced neutrophil recruitment in the bronchoalveolar space by more than 64%. Moreover, functional inhibition of LFA-1 and Mac-1 abolished CLP-induced lung damage and edema. However, formation of CXC chemokines in the lung was intact in mice pretreated with the anti-LFA-1 and anti-Mac-1 antibodies. Our data demonstrate that both LFA-1 and Mac-1 regulate pulmonary infiltration of neutrophils and lung edema associated with abdominal sepsis. Thus, these novel findings suggest that LFA-1 or Mac-1 may serve as targets to protect against lung injury in polymicrobial sepsis.

Hepatic infiltration of activated CD8 lymphocytes is a major feature of graft-vs-host disease (GvHD). Chemoattractant cytokines and their receptors are key regulators of lymphocyte trafficking, but the involvement of chemoattractant receptors in the physiologic recruitment of cells into the inflamed liver has not been defined. The present study examines the role of the chemokine receptor CXCR6, which is highly expressed by liver-infiltrating CD8 T cells. Hepatic accumulation of donor CD8, but not donor CD4, lymphocytes was significantly reduced in GvHD induced by transfer of CXCR6−/−, H-2Db lymphocytes into BDF1, H-2Dbxd recipients. To determine whether altered recruitment contributes to the reduced accumulation, CXCR6−/− or wild-type splenic lymphocytes participating in an active GvHD response were isolated and transferred i.v. into secondary recipients with active GvHD, and the short term (6-h) recruitment of transferred cells to the inflamed liver was assessed. CXCR6−/− CD8 (but not CD4) cells displayed a significant (33%) reduction in liver localization, whereas frequencies in blood of CXCR6−/− and wild-type CD8 cells were similar. Proliferation and apoptosis of liver-infiltrating donor CD8 cells were unaffected. We conclude that CXCR6 helps mediate the recruitment of activated CD8 lymphocytes in GvHD-induced hepatitis and may be a useful target to treat pathological inflammation in the liver.

Abdominal sepsis is associated with dysfunctional hemostasis. Thrombin generation (TG) is a rate-limiting step in systemic coagulation. Neutrophils can expell neutrophil extracellular traps (NETs) and/or microparticles (MPs) although their role in pathological coagulation remains elusive. Cecal ligation and puncture (CLP)-induced TG in vivo was reflected by a reduced capacity of plasma from septic animals to generate thrombin. Depletion of neutrophils increased TG in plasma from CLP mice. Sepsis was associated with increased histone 3 citrullination in neutrophils and plasma levels of cell-free DNA and DNA-histone complexes and administration of DNAse not only eliminated NET formation but also elevated TG in sepsis. Isolated NETs increased TG and co-incubation with DNAse abolished NET-induced formation of thrombin. TG triggered by NETs was inhibited by blocking factor XII and abolished in factor XII-deficient plasma but intact in factor VII-deficient plasma. Activation of neutrophils simultaneously generated large amount of neutrophil-derived MPs, which were found to bind to NETs via histone-phosphatidylserine interactions. These findings show for the first time that NETs and MPs physically interact, and that NETs might constitute a functional assembly platform for MPs. We conclude that NET-MP complexes induce TG via the intrinsic pathway of coagulation and that neutrophil-derived MPs play a key role in NET-dependent coagulation.

Excessive neutrophil activation is a major component in septic lung injury. Neutrophil-derived DNA may form extracellular traps in response to bacterial invasions. The aim of the present study was to investigate the potential role of neutrophil extracellular traps (NETs) in septic lung injury. Male C57BL/6 mice were treated with recombinant human (rh)DNAse (5 mg/kg) after cecal ligation and puncture (CLP). Extracellular DNA was stained by Sytox green, and NET formation was quantified by confocal microscopy and cell-free DNA in plasma, peritoneal cavity, and lung. Blood, peritoneal fluid, and lung tissue were harvested for analysis of neutrophil infiltration, NET levels, tissue injury, as well as CXC chemokine and cytokine formation. We observed that CLP caused increased formation of NETs in plasma, peritoneal cavity, and lung. Administration of rhDNAse not only eliminated NET formation in plasma, peritoneal cavity, and bronchoalveolar space but also reduced lung edema and tissue damage 24 h after CLP induction. Moreover, treatment with rhDNAse decreased CLP-induced formation of CXC chemokines, IL-6, and high-mobility group box 1 (HMGB1) in plasma, as well as CXC chemokines and IL-6 in the lung. In vitro, we found that neutrophil-derived NETs had the capacity to stimulate secretion of CXCL2, TNF-α, and HMGB1 from alveolar macrophages. Taken together, our findings show that NETs regulate pulmonary infiltration of neutrophils and tissue injury via formation of proinflammatory compounds in abdominal sepsis. Thus we conclude that NETs exert a proinflammatory role in septic lung injury.