Ischemia reperfusion (IR)-induced oxidative stress and inflammation contribute to morbidity and mortality of acute coronary syndrome. Ischemia results in profound hypoxia and tissue dysfunction and subsequent reperfusion further aggravates ischemic cardiac tissue damage. In cardiac IR injury, neutrophils are involved both in causing cardiomyocyte death and in preserving heart tissue homeostasis. We tested the hypothesis that neutrophil subpopulations show distinct functions in the pathogenesis of cardiac IR injury and that their functional heterogeneity can be exploited in subset-specific pharmacological intervention to prevent IR-induced myocardial tissue damage and functional deterioration. Cardiac IR-injury in a mouse model was characterized by the presence of two distinct heart-inflammatory subsets of neutrophils, one that specifically endocytosed albumin nanoparticles (ANPhigh) and one that endocytose few or no ANP (ANPlow). The two subsets had very distinct inflammatory phenotypes. ANPhigh neutrophils expressed significantly greater amounts of inflammatory mediators, such as Il-1b and Ccl3, than ANPlow neutrophils. Targeting the Spleen tyrosine kinase (Syk) specifically in ANPhigh neutrophils post IR reduced cardiac neutrophilic and mononuclear inflammation and drastically decreased infarct size, and prevented the deterioration of cardiac function. Targeting the Syk pathway specifically in a defined subset of neutrophils is a feasible therapy for cardiac IR injury.

Dysregulated neutrophil infiltration and activity in lung tissues are pathogenic features of sepsis-induced acute respiratory distress syndrome (ARDS), an important cause of morbidity and mortality in critically ill patients. Therapeutic reduction of excessive neutrophil transmigration from the vasculature into the distal airspaces to mitigate lung tissue damage and improve survival is an important objective. Human peripheral blood polymorphonuclear neutrophils (PMNs) can be separated into two subsets as recently described: one that readily endocytoses albumin nanoparticles (ANPs) referred to as ANP(high) PMN and another that fails to endocytose ANPs referred to as ANP(low) PMN. Here, we tested the hypothesis that targeting ANP(high) human PMNs with ANPs loaded with the drug piceatannol (PANPs), a selective spleen tyrosine kinase (Syk) inhibitor that inhibits beta 2-integrin signaling, would mitigate lipopolysaccharide (LPS)-induced accumulation of PMNs in human lungs and reduce lung edema. In the present study, human lungs were perfused ex vivo with fresh whole blood containing human PMNs and 6 mg of LPS to induce injury accompanied by PMN infiltration into the lungs. Following the LPS exposure, intravenous injection of PANPs reduced transmigration and accumulation of PMNs in the lungs as determined by bronchoalveolar lavage counts and histologic scoring. A parallel reduction in lung weight gain, a surrogate for pulmonary edema fluid accumulation, suggested protection from edema in this human lung injury model. The results demonstrate the potential of therapeutic drug loading of albumin nanoparticles (PANPs) for targeting an injurious, inflammation-inducing population of PMNs in ARDS patients.

The complex involvement of neutrophils in inflammatory diseases makes them intriguing but challenging targets for therapeutic intervention. Here, we tested the hypothesis that varying endocytosis capacities would delineate functionally distinct neutrophil subpopulations that could be specifically targeted for therapeutic purposes. By using uniformly sized (∼120 nm in diameter) albumin nanoparticles (ANP) to characterize mouse neutrophils in vivo, we found two subsets of neutrophils, one that readily endocytosed ANP (ANPhigh neutrophils) and another that failed to endocytose ANP (ANPlow population). These ANPhigh and ANPlow subsets existed side by side simultaneously in bone marrow, peripheral blood, spleen, and lungs, both under basal conditions and after inflammatory challenge. Human peripheral blood neutrophils showed a similar duality. ANPhigh and ANPlow neutrophils had distinct cell surface marker expression and transcriptomic profiles, both in naive mice and in mice after endotoxemic challenge. ANPhigh and ANPlow neutrophils were functionally distinct in their capacities to kill bacteria and to produce inflammatory mediators. ANPhigh neutrophils produced inordinate amounts of reactive oxygen species and inflammatory chemokines and cytokines. Targeting this subset with ANP loaded with the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, mitigated the effects of polymicrobial sepsis by reducing tissue inflammation while fully preserving neutrophilic host-defense function.

The pathogenesis of lung fibrosis involves hyperactivation of innate and adaptive immune pathways that release inflammatory cytokines and growth factors such as tumor growth factor (TGF)β1 and induce aberrant extracellular matrix protein production. During the genesis of pulmonary fibrosis, resident alveolar macrophages are replaced by a population of newly arrived monocyte-derived interstitial macrophages that subsequently transition into alveolar macrophages (Mo-AMs). These transitioning cells initiate fibrosis by releasing profibrotic cytokines and remodeling the matrix. Here, we describe a strategy for leveraging the up-regulation of the mannose receptor CD206 in interstitial macrophages and Mo-AM to treat lung fibrosis. We engineered mannosylated albumin nanoparticles, which were found to be internalized by fibrogenic CD206+ monocyte derived macrophages (Mo-Macs). Mannosylated albumin nanoparticles incorporating TGFβ1 small-interfering RNA (siRNA) targeted the profibrotic subpopulation of CD206+ macrophages and prevented lung fibrosis. The findings point to the potential utility of mannosylated albumin nanoparticles in delivering TGFβ-siRNA into CD206+ profibrotic macrophages as an antilung fibrosis strategy.

Polymorphonuclear neutrophils (PMN) are highly dynamic innate immune cells which are essential for lung host defense. However, in vivo intravital imaging in moving organs such as the lung remains challenging due to motion artifacts. Here we describe a novel intravital imaging method with high-throughput analytical capability based on a computer vision stabilization algorithm, Computer-vision-Assisted STabilized intravital imaging (CASTii). The sub-micron precision of this approach enables analysis of compartmentalized intravital PMN dynamics. We quantified in real-time a novel patrolling function of lung intracapillary circulating PMN. We also describe the dynamics of intracapillary PMN pooling (marginated PMN pool) using direct imaging of PMNs. The pool was formed by repeated catch-and-release kinetics involving PMN deformation inside microvessels during the passage of PMNs in vessels. We observed rapid PMN recruitment into the lung tissue compartments from pooled PMNs in response to alveolar chemoattract exposure. In contrast, endotoxemia-induced intracapillary sequestration of PMN impaired PMN transmigration into the alveolar space and defective phagocytosis of live bacteria. Intravital imaging of PMN dynamics with CASTii provides fundamental insights into host-defense functions of lung capillary PMN.

Inflammatory tissue injury such as acute lung injury (ALI) is a disorder that leads to respiratory failure, a major cause of morbidity and mortality worldwide. Excessive neutrophil influx is a critical pathogenic factor in the development of ALI. Here, we identify the subset of neutrophils that is responsible for ALI and lethality in polymicrobial sepsis. The pro-inflammatory neutrophil subpopulation was characterized by its unique ability to endocytose albumin nanoparticles (ANP), upregulation of pro-inflammatory cytokines and chemokines as well as the excessive production of reactive oxygen species (ROS) in models of endotoxemia and septicemia. ANP delivery of the drug piceatannol, a spleen tyrosine kinase (Syk) inhibitor, to the susceptible subset of neutrophils, prevented ALI and mortality in mice subjected to polymicrobial infection. Targeted inhibition of Syk in ANP-susceptible neutrophils had no detrimental effect on neutrophil-dependent host defense because the subset of ANP low neutrophils effectively controlled polymicrobial infection. The results show that neutrophil heterogeneity can be leveraged therapeutically to prevent ALI without compromising host defense.

The transcriptional repressor DREAM is involved in pain sensing. Tiruppathi and colleagues show that it is also involved in innate signaling by regulating the anti-inflammatory deubiquitinase A20. Here we found that the transcription repressor DREAM bound to the promoter of the gene encoding A20 to repress expression of this deubiquitinase that suppresses inflammatory NF-κB signaling. DREAM-deficient mice displayed persistent and unchecked A20 expression in response to endotoxin. DREAM functioned by transcriptionally repressing A20 through binding to downstream regulatory elements (DREs). In contrast, binding of the transcription factor USF1 to the DRE-associated E-box domain in the gene encoding A20 activated its expression in response to inflammatory stimuli. Our studies define the critical opposing functions of DREAM and USF1 in inhibiting and inducing A20 expression, respectively, and thereby the strength of NF-κB signaling. Targeting of DREAM to induce USF1-mediated A20 expression is therefore a potential anti-inflammatory strategy for the treatment of diseases associated with unconstrained NF-κB activity, such as acute lung injury.

Myocarditis, often due to an aberrant immune response to infection, is a major cause of dilated cardiomyopathy. Microbial pattern recognition receptors, such as TLRs, orchestrate the cytokine and chemokine responses that augment or limit the severity of myocarditis. Using the mouse model of experimental autoimmune myocarditis (EAM), in which disease is induced by immunization with a heart-specific self peptide and the agonist to multiple TLRs, complete Freund's adjuvant, we found that increased serum concentrations of the chemokine CXCL1/KC correlated directly with decreased severity of myocarditis. To directly test whether CXCL1/KC caused the amelioration of myocarditis, we treated mice, after challenge with heart-specific self peptide, with exogenous recombinant CXCL1/KC. We found that the administration of recombinant mouse CXCL1/KC completely abrogated heart inflammatory infiltration and cardiomyocyte damage. Moreover, we show that TLR4 signaling is required to increase serum protein concentrations of CXCL1/KC in EAM, and we demonstrate that the administration of the TLR4 agonist LPS significantly decreased severity and prevalence of EAM and reduced the number of heart-specific self peptide reactive effector T cells. These findings reveal a novel function of CXCL1/KC in the context of organ-specific autoimmune disease that may prove useful for the treatment of inflammatory conditions that underlie human heart disease.

Heart diseases are the most prevalent cause of morbidity and mortality in rich countries. A common cause of progressive heart disease, heart failure, and sudden death is dilated cardiomyopathy (DCM), a group of disorders in which the heart muscle is weakened and cannot pump effectively. Chlamydia infections, in particular, are epidemiologically linked to human heart disease. Chlamydia infections cause pneumonia and conjunctivitis in children and are a primary cause of sexually transmitted diseases and female infertility. The development of a murine model of autoimmune myocarditis was based on genetic differences among inbred mouse strains in the immune response to CVB3-induced myocarditis. Experimental induction of most autoimmune diseases requires CD4+ T-helper cells responsive to the self-antigen presented on major histocompatability complex (MHC) class II molecules, and administration of monoclonal antibodies (MAbs) against CD4 molecules can block disease in experimental and genetic animal models of autoimmunity. Hence, CD4+ T-helper lymphocytes appear to be crucial regulators for the initiation of T-cell-dependent autoimmunity, and clinical trials that target the CD4+ cell lineage are under way in an examination of treatments for various human autoimmune diseases. Chronic heart diseases in humans have been linked to certain HLA alleles, such as HLA-DQ6. Experimental in vivo and in vitro data provide evidence of molecular mimicry between bacterial antigens and heart-specific proteins and indicate that bacterial peptides can trigger tissue-specific inflammation of the heart. After initiation of the inflammatory process by CD4+ T cells, CD8+ cells and macrophages are recruited into the heart muscle and contribute to disease pathogenesis.

The complement anaphylatoxin C5a has a pathogenetic role in endotoxin-induced lung inflammatory injury by regulating phagocytic cell migration and activation. Endotoxin and C5a activate the enzyme sphingosine kinase (Sphk) 1 to generate the signaling lipid sphingosine-1-phosphate (S1P), a critical regulator of phagocyte function. We assessed the function of Sphk1 and S1P in experimental lung inflammatory injury and determined their roles in anaphylatoxin receptor signaling and on the expression of the two C5a receptors, C5aR (CD88) and C5L2, on phagocytes. We report that Sphk1 gene deficient (Sphk1(-/-)) mice had augmented lung inflammatory response to endotoxin compared to wild type mice. Sphk1 was required for C5a-mediated reduction in cytokine and chemokine production by macrophages. Moreover, neutrophils from Sphk1(-/-) mice failed to upregulate the anaphylatoxin receptor C5L2 in response to LPS. Exogenous S1P restored C5L2 cell surface expression of Sphk1(-/-) mouse neutrophils to wild type levels but had no effect on cell surface expression of the other anaphylatoxin receptor, CD88. These results provide the first genetic evidence of the crucial role of Sphk1 in regulating the balance between expression of CD88 and C5L2 in phagocytes. S1P-mediated up-regulation of C5L2 is a novel therapeutic target for mitigating endotoxin-induced lung inflammatory injury.

Human embryonic stem cells differentiated under mesoderm-inducing conditions have important therapeutic properties in sepsis-induced lung injury in mice. Single cell suspensions obtained from day 7 human embryoid bodies (d7EBs) injected i.v. 1 hour after cecal ligation and puncture significantly reduced lung inflammation and edema as well as production of tumor necrosis factor-α and interferon-γ in lungs compared with controls, whereas interleukin-10 production remained elevated. d7EB cell transplantation also reduced mortality to 50% from 90% in the control group. The protection was ascribed to d7EB cell interaction with lung resident CD11b+ cells, and was correlated with the ability of d7EB cells to reduce it also reduced production of proinflammatory cytokines by CD11+ cells, and to endothelial NO synthase-derived NO by d7EB cells, leading to inhibition of inducible macrophage-type NO synthase activation in CD11b+ cells. The protective progenitor cells were positive for the endothelial and hematopoietic lineage marker angiotensin converting enzyme (ACE). Only the ACE+ fraction modulated the proinflammatory profile of CD11b+ cells and reduced mortality in septic mice. In contrast to the nonprotective ACE-cell fraction, the ACE+ cell fraction also produced NO. These findings suggest that an ACE+ subset of human embryonic stem cell-derived progenitor cells has a highly specialized anti-inflammatory function that ameliorates sepsis-induced lung inflammation and reduces mortality.

The mechanism underlying the protective effect of sphingosine kinase 1 (SphK1) in inflammatory injury is not clear. We demonstrated using SphK1-null mice (SphK1(-/-)) the crucial role of SphK1 in suppressing lipopolysaccharide-induced neutrophil oxidant production and sequestration in lungs and mitigating lung inflammatory injury. This effect of SphK1 was independent of the production of sphingosine 1-phosphate, the product of SphK1 activity. The anti-inflammatory effect of SphK1 in the lipopolysaccharide model was mediated through SphK1 interaction with JNK. SphK1 stabilization of JNK in turn inhibited JNK binding to the JNK-interacting protein 3 (JIP3) and thus abrogated the activation of NADPH oxidase and oxidant generation and resultant NF-kappa B activation. Therefore, SphK1-mediated down-regulation of JNK activity serves to dampen inflammation and tissue injury.

Activation of NF-kappaB is essential for protease-activated receptor-1 (PAR-1)-mediated ICAM-1 expression in endothelial cells. Here we show that PAR-1 activation induces binding of both p65/RelA and NFATc1 to the NF-kappaB binding site localized in intron-1 of the ICAM-1 gene to initiate transcription in endothelial cells. We discovered the presence of two NF-kappaB binding sites in intron-1 (+70, NF-kappaB site 1; +611, NF-kappaB site 2) of the human ICAM-1 gene. Chromatin immunoprecipitation results showed that thrombin induced binding of p65/RelA and of NFATc1 specifically to intronic NF-kappaB site 1 of the ICAM-1 gene. Electrophoretic mobility shift and supershift assays confirmed the binding of p65/RelA and NFATc1 to the intronic NF-kappaB site 1 in thrombin-stimulated cells. Thrombin increased the expression of ICAM-1-promoter-intron 1-reporter (-1,385 to +234) construct approximately 25-fold and mutation of intronic NF-kappaB site 1 markedly reduced thrombin-induced reporter expression. Moreover, inhibition of calcineurin, knockdown of either NFATc1 or p65/RelA with siRNA significantly reduced thrombin-induced ICAM-1 expression and polymorphonuclear leukocyte adhesion to endothelial cells. In contrast, NFATc1 knockdown had no effect on TNF-alpha-induced ICAM-1 expression. Thus these results suggest that p65/RelA and NFATc1 bind to the intronic NF-kappaB site 1 sequence to induce optimal transcription of the ICAM-1 gene in response to thrombin in endothelial cells.

Airway inflammation is a common feature of cystic fibrosis (CF) lung disease. Major histocompatibility complex class II (MHCII) molecules expressed on dendritic cells (DCs) play a pivotal role in presenting bacterial antigens to T lymphocytes to trigger the immune response. We show here that the cystic fibrosis transmembrane regulator (CFTR) is expressed in DCs where it participates in regulating plasma membrane MHCII expression. DCs from CFTR−/− mice showed enhanced plasma membrane expression of MHCII as well as stimulatory effect on CD4+ T cell proliferation. Metabolic labeling studies showed that degradation of invariant chain (Ii) in MHCII‐Ii complex was significantly greater in DCs from CFTR−/− mice than CFTR+/+ mice. Lysosomal acidification in DCs from CFTR−/− mice was also greater and acidification was prevented by amiloride, an epithelial sodium channel (ENaC) inhibitor. Thus, CFTR may regulate MHCII plasma membrane expression and antigen presentation by inhibiting lysosomal ENaC, and thus modulate lysosomal acidification. These results suggest a novel pathogenic mechanism of inflammation in cystic fibrosis patients based on the hyper‐activation of the immune response. This work was supported by Parker B. Francis Fellowships in Pulmonary Research and US National Institutes of Health grants P01 HL77806.

The E3 ubiquitin ligase Cblb has a crucial role in the prevention of chronic inflammation and autoimmunity. Here we show that Cblb also has an unexpected function in acute lung inflammation. Cblb attenuates the sequestration of inflammatory cells in the lungs after administration of lipopolysaccharide (LPS). In a model of polymicrobial sepsis in which acute lung inflammation depends on the LPS receptor (Toll-like receptor 4, TLR-4), the loss of Cblb expression accentuates acute lung inflammation and reduces survival. Loss of Cblb significantly increases sepsis-induced release of inflammatory cytokines and chemokines. Cblb controls the association between TLR4 and the intracellular adaptor MyD88. Expression of wild-type Cblb, but not expression of a Cblb mutant that lacks E3 ubiquitin ligase function, prevents the activity of a reporter gene for the transcription factor nuclear factor-κB (NF-κB) in monocytes that have been challenged with LPS. The downregulation of TLR4 expression on the cell surface of neutrophils is impaired in the absence of Cblb. Our data reveal that Cblb regulates the TLR4-mediated acute inflammatory response that is induced by sepsis.

Chlamydial infections are among the most common human infections. Every year, in millions of humans, they cause infections of the eyes, the respiratory tract, the genital tract, joints, and the vasculature. Chlamydiae are obligate intracellular prokaryotic pathogens. Chlamydiae promote, in susceptible host cells that include mucosal epithelial cells, vascular endothelial cells, smooth muscle cells, and monocytes and macrophages, their survival while causing disease of varying clinical importance and consequence in their hosts. Chlamydia infections often precede the initiation of autoimmune diseases, and Chlamydiae are often found within autoimmune lesions. Thus, they have been suspected in the etiology and pathogenesis of autoimmune diseases. Autoimmune diseases have many causes. Genes, notably genes encoding cell-surface proteins that display peptides for immune recognition, the major histocompatibility complex (MHC), the environment, and the microbial diversity within the human body determine the susceptibility to autoimmune diseases. One mechanism by which infection is linked to the initiation of autoimmunity is termed molecular mimicry. Molecular mimicry describes the phenomenon of protein products from dissimilar genes sharing similar structures that elicit an immune response to both self and microbial proteins. Molecular mimicry might thus be a mechanism by which infections trigger autoimmune diseases. For the purpose of this chapter, we will focus on chlamydial proteins that mimic host self-proteins and thus contribute to initiation and maintenance of autoimmune diseases. Thus far, the strongest cases for molecular mimicry seem to have been made for chlamydial heat shock proteins 60, the DNA primase of Chlamydia trachomatis, and chlamydial OmcB proteins.