The Histone Methyltransferase Setdb2 Modulates Macrophage Phenotype and Uric Acid Production in Diabetic Wound Repair.

Macrophage plasticity is critical for normal tissue repair to ensure transition from the inflammatory to the proliferative phase of healing. We examined macrophages isolated from wounds of patients afflicted with diabetes and of healthy controls and found differential expression of the methyltransferase Setdb2. Myeloid-specific deletion of Setdb2 impaired the transition of macrophages from an inflammatory phenotype to a reparative one in normal wound healing. Mechanistically, Setdb2 trimethylated histone 3 at NF-κB binding sites on inflammatory cytokine gene promoters to suppress transcription. Setdb2 expression in wound macrophages was regulated by interferon (IFN) ß, and under diabetic conditions, this IFNß-Setdb2 axis was impaired, leading to a persistent inflammatory macrophage phenotype in diabetic wounds. Setdb2 regulated the expression of xanthine oxidase and thereby the uric acid (UA) pathway of purine catabolism in macrophages, and pharmacologic targeting of Setdb2 or the UA pathway improved healing. Thus, Setdb2 regulates macrophage plasticity during normal and pathologic wound repair and is a target for therapeutic manipulation.

Dysregulation of intercellular signaling by MOF deletion leads to liver injury.

Epigenetic mechanisms that alter heritable gene expression and chromatin structure play an essential role in many biological processes, including liver function. Human MOF (males absent on the first) is a histone acetyltransferase that is globally downregulated in human steatohepatitis. However, the function of MOF in the liver remains unclear. Here, we report that MOF plays an essential role in adult liver. Genetic deletion of Mof by Mx1-Cre in the liver leads to acute liver injury, with increase of lipid deposition and fibrosis akin to human steatohepatitis. Surprisingly, hepatocyte-specific Mof deletion had no overt liver abnormality. Using the in vitro coculturing experiment, we show that Mof deletion-induced liver injury requires coordinated changes and reciprocal signaling between hepatocytes and Kupffer cells, which enables feedforward regulation to augment inflammation and apoptotic responses. At the molecular level, Mof deletion induced characteristic changes in metabolic gene programs, which bore noticeable similarity to the molecular signature of human steatohepatitis. Simultaneous deletion of Mof in both hepatocytes and macrophages results in enhanced expression of inflammatory genes and NO signaling in vitro. These changes, in turn, lead to apoptosis of hepatocytes and lipotoxicity. Our work highlights the importance of histone acetyltransferase MOF in maintaining metabolic liver homeostasis and sheds light on the epigenetic dysregulation in liver pathogenesis.

Upregulation of H3K27 Demethylase KDM6 During Respiratory Syncytial Virus Infection Enhances Proinflammatory Responses and Immunopathology.

Severe disease following respiratory syncytial virus (RSV) infection has been linked to enhanced proinflammatory cytokine production that promotes a Th2-type immune environment. Epigenetic regulation in immune cells following viral infection plays a role in the inflammatory response and may result from upregulation of key epigenetic modifiers. In this study, we show that RSV-infected bone marrow-derived dendritic cells (BMDC) as well as pulmonary dendritic cells (DC) from RSV-infected mice upregulated the expression of Kdm6b/Jmjd3 and Kdm6a/Utx, H3K27 demethylases. KDM6-specific chemical inhibition (GSK J4) in BMDC led to decreased production of chemokines and cytokines associated with the inflammatory response during RSV infection (i.e., CCL-2, CCL-3, CCL-5, IL-6) as well as decreased MHC class II and costimulatory marker (CD80/86) expression. RSV-infected BMDC treated with GSK J4 altered coactivation of T cell cytokine production to RSV as well as a primary OVA response. Airway sensitization of naive mice with RSV-infected BMDCs exacerbate a live challenge with RSV infection but was inhibited when BMDCs were treated with GSK J4 prior to sensitization. Finally, in vivo treatment with the KDM6 inhibitor, GSK J4, during RSV infection reduced inflammatory DC in the lungs along with IL-13 levels and overall inflammation. These results suggest that KDM6 expression in DC enhances proinflammatory innate cytokine production to promote an altered Th2 immune response following RSV infection that leads to more severe immunopathology.

Sepsis Induces Prolonged Epigenetic Modifications in Bone Marrow and Peripheral Macrophages Impairing Inflammation and Wound Healing.

OBJECTIVE: Sepsis represents an acute life-threatening disorder resulting from a dysregulated host response. For patients who survive sepsis, there remains long-term consequences, including impaired inflammation, as a result of profound immunosuppression. The mechanisms involved in this long-lasting deficient immune response are poorly defined. Approach and Results: Sepsis was induced using the murine model of cecal ligation and puncture. Following a full recovery period from sepsis physiology, mice were subjected to our wound healing model and wound macrophages (CD11b+, CD3-, CD19-, Ly6G-) were sorted. Post-sepsis mice demonstrated impaired wound healing and decreased reepithelization in comparison to controls. Further, post-sepsis bone marrow-derived macrophages and wound macrophages exhibited decreased expression of inflammatory cytokines vital for wound repair (IL [interleukin]-1ß, IL-12, and IL-23). To evaluate if decreased inflammatory gene expression was secondary to epigenetic modification, we conducted chromatin immunoprecipitation on post-sepsis bone marrow-derived macrophages and wound macrophages. This demonstrated decreased expression of Mll1, an epigenetic enzyme, and impaired histone 3 lysine 4 trimethylation (activation mark) at NFκB (nuclear factor kappa-light-chain-enhancer of activated B cells)-binding sites on inflammatory gene promoters in bone marrow-derived macrophages and wound macrophages from postcecal ligation and puncture mice. Bone marrow transplantation studies demonstrated epigenetic modifications initiate in bone marrow progenitor/stem cells following sepsis resulting in lasting impairment in peripheral macrophage function. Importantly, human peripheral blood leukocytes from post-septic patients demonstrate a significant reduction in MLL1 compared with nonseptic controls. CONCLUSIONS: These data demonstrate that severe sepsis induces stable mixed-lineage leukemia 1-mediated epigenetic modifications in the bone marrow, which are passed to peripheral macrophages resulting in impaired macrophage function and deficient wound healing persisting long after sepsis recovery.

The Role of Iron in the Susceptibility of Neonatal Mice to Escherichia coli K1 Sepsis.

Sepsis from Escherichia coli expressing the K1 antigen is a leading cause of death in neonates. In a murine model, E. coli K1 grew rapidly in the peritoneal cavity of neonatal mice, causing fatal disease. In contrast, adult mice cleared the infection. Neonatal mice mounted a rapid and equivalent antimicrobial immune response compared to adult mice. Interestingly, peritoneal fluid from neonatal mice contained significantly more total iron than that of adult mice, which was sufficient to support enhanced E. coli growth. Transient iron overload in adult mice infected with E. coli resulted in 100% mortality. Maternal diet-induced mild iron deficiency decreased offspring peritoneal iron, decreased bacterial growth, and conferred protection against sepsis. Taken together, neonatal susceptibility to E. coli K1 sepsis is enhanced by a localized excess of peritoneal iron that allows for unchecked bacterial growth. Targeting this excess iron may provide a new therapeutic target in human patients.

Hox5 Paralogous Genes Modulate Th2 Cell Function during Chronic Allergic Inflammation via Regulation of Gata3.

Allergic asthma is a significant health burden in western countries, and continues to increase in prevalence. Th2 cells contribute to the development of disease through release of the cytokines IL-4, IL-5, and IL-13, resulting in increased airway eosinophils and mucus hypersecretion. The molecular mechanisms behind the disease pathology remain largely unknown. In this study we investigated a potential regulatory role for the Hox5 gene family, Hoxa5, Hoxb5, and Hoxc5, genes known to be important in lung development within mesenchymal cell populations. We found that Hox5-mutant mice show exacerbated pathology compared with wild-type controls in a chronic allergen model, with an increased Th2 response and exacerbated lung tissue pathology. Bone marrow chimera experiments indicated that the observed enhanced pathology was mediated by immune cell function independent of mesenchymal cell Hox5 family function. Examination of T cells grown in Th2 polarizing conditions showed increased proliferation, enhanced Gata3 expression, and elevated production of IL-4, IL-5, and IL-13 in Hox5-deficient T cells compared with wild-type controls. Overexpression of FLAG-tagged HOX5 proteins in Jurkat cells demonstrated HOX5 binding to the Gata3 locus and decreased Gata3 and IL-4 expression, supporting a role for HOX5 proteins in direct transcriptional control of Th2 development. These results reveal a novel role for Hox5 genes as developmental regulators of Th2 immune cell function that demonstrates a redeployment of mesenchyme-associated developmental genes.

Notch Ligand Delta-like 4 Promotes Regulatory T Cell Identity in Pulmonary Viral Infection.

Regulatory T (Treg) cells establish tolerance, prevent inflammation at mucosal surfaces, and regulate immunopathology during infectious responses. Recent studies have shown that Delta-like ligand 4 (Dll4) was upregulated on APC after respiratory syncytial virus (RSV) infection, and its inhibition leads to exaggerated immunopathology. In the present study, we outline the role of Dll4 in Treg cell differentiation, stability, and function in RSV infection. We found that Dll4 was expressed on CD11b+ pulmonary dendritic cells in the lung and draining lymph nodes in wild-type BALB/c mice after RSV infection. Dll4 neutralization exacerbated RSV-induced disease pathology, mucus production, group 2 innate lymphoid cell infiltration, IL-5 and IL-13 production, as well as IL-17A+ CD4 T cells. Dll4 inhibition decreased the abundance of CD62LhiCD44loFoxp3+ central Treg cells in draining lymph nodes. The RSV-induced disease was accompanied by an increase in Th17-like effector phenotype in Foxp3+ Treg cells and a decrease in granzyme B expression after Dll4 blockade. Finally, Dll4-exposed induced Treg cells maintained the CD62LhiCD44lo central Treg cell phenotype, had increased Foxp3 expression, became more suppressive, and were resistant to Th17 skewing in vitro. These results suggest that Dll4 activation during differentiation sustained Treg cell phenotype and function to control RSV infection.

The STAT4/MLL1 Epigenetic Axis Regulates the Antimicrobial Functions of Murine Macrophages.

Macrophages are critical immune cells for the clearance of microbial pathogens and cellular debris from peripheral tissues. Macrophage inflammatory responses are governed by gene expression patterns, and these patterns are often subject to epigenetic control. Chromatin modifications, such as histone methylation, regulate gene accessibility in macrophages, and macrophage polarization is governed in part by the expression and function of chromatin-modifying enzymes. The histone methyltransferase mixed-lineage leukemia 1 (MLL1) preferentially modifies lysine residue 4 on the unstructured protein tail of histone H3. MLL1 expression and function have been shown to be governed by signal transduction pathways that are activated by inflammatory stimuli, such as NF-κB. Therefore, we sought to investigate the role of MLL1 in mediating macrophage inflammatory responses. Bone marrow-derived macrophages from mice with a targeted MLL1 gene knockout (Lys2-Cre+/- MLL1fx/fx) exhibited decreased proinflammatory gene expression with concurrent decreases in activating histone methylation. However, MLL1-deficient macrophages also exhibited increased phagocytic and bacterial killing activity in vitro. RNA profiling of MLL1-knockout macrophages identified numerous genes involved with inflammatory responses whose expression was altered in response to TLR ligands or proinflammatory cytokines, including STAT4. STAT4-dependent cytokines, such as type I IFNs were able to drive MLL1 expression in macrophages, and MLL1-knockout macrophages exhibited decreased activating histone methylation in the STAT4 promoter. These results implicate an important role for MLL1-dependent epigenetic regulation of macrophage antimicrobial functions.

Type I Interferon Induced Epigenetic Regulation of Macrophages Suppresses Innate and Adaptive Immunity in Acute Respiratory Viral Infection.

Influenza A virus (IAV) is an airborne pathogen that causes significant morbidity and mortality each year. Macrophages (Mϕ) are the first immune population to encounter IAV virions in the lungs and are required to control infection. In the present study, we explored the mechanism by which cytokine signaling regulates the phenotype and function of Mϕ via epigenetic modification of chromatin. We have found that type I interferon (IFN-I) potently upregulates the lysine methyltransferase Setdb2 in murine and human Mϕ, and in turn Setdb2 regulates Mϕ-mediated immunity in response to IAV. The induction of Setdb2 by IFN-I was significantly impaired upon inhibition of the JAK-STAT signaling cascade, and chromatin immunoprecipitation revealed that both STAT1 and interferon regulatory factor 7 bind upstream of the transcription start site to induce expression. The generation of Setdb2LacZ reporter mice revealed that IAV infection results in systemic upregulation of Setdb2 in myeloid cells. In the lungs, alveolar Mϕ expressed the highest level of Setdb2, with greater than 70% lacZ positive on day 4 post-infection. Silencing Setdb2 activity in Mϕ in vivo enhanced survival in lethal IAV infection. Enhanced host protection correlated with an amplified antiviral response and less obstruction to the airways. By tri-methylating H3K9, Setdb2 silenced the transcription of Mx1 and Isg15, antiviral effectors that inhibit IAV replication. Accordingly, a reduced viral load in knockout mice on day 8 post-infection was linked to elevated Isg15 and Mx1 transcript in the lungs. In addition, Setdb2 suppressed the expression of a large number of other genes with proinflammatory or immunomodulatory function. This included Ccl2, a chemokine that signals through CCR2 to regulate monocyte recruitment to infectious sites. Consistently, knockout mice produced more CCL2 upon IAV infection and this correlated with a 2-fold increase in the number of inflammatory monocytes and alveolar Mϕ in the lungs. Finally, Setdb2 expression by Mϕ suppressed IL-2, IL-10, and IFN-γ production by CD4+ T cells in vitro, as well as proliferation in IAV-infected lungs. Collectively, these findings identify Setdb2 as a novel regulator of the immune system in acute respiratory viral infection.

Sirtuin 1 Regulates Dendritic Cell Activation and Autophagy during Respiratory Syncytial Virus-Induced Immune Responses.

Respiratory syncytial virus (RSV) is the major cause of lower respiratory tract infection in children worldwide. Sirtuin 1 (SIRT1), an NAD(+)-dependent deacetylase, has been associated with the induction of autophagy and the regulation of inflammatory mediators. We found that Sirt1 was upregulated in mouse lung after RSV infection. Infected animals that received EX-527, a selective SIRT1 inhibitor, displayed exacerbated lung pathology, with increased mucus production, elevated viral load, and enhanced Th2 cytokine production. Gene expression analysis of isolated cell populations revealed that Sirt1 was most highly upregulated in RSV-treated dendritic cells (DCs). Upon RSV infection, EX-527-treated DCs, Sirt1 small interfering RNA-treated DCs, or DCs from conditional knockout (Sirt1(f/f)-CD11c-Cre(+)) mice showed downregulated inflammatory cytokine gene expression and attenuated autophagy. Finally, RSV infection of Sirt1(f/f)-CD11c-Cre(+) mice resulted in altered lung and lymph node cytokine responses, leading to exacerbated pathology. These data indicate that SIRT1 promotes DC activation associated with autophagy-mediated processes during RSV infection, thereby directing efficient antiviral immune responses.

IL-17RB+ granulocytes are associated with airflow obstruction in asthma.

BACKGROUND: Interleukin (IL)-25 (IL-17E) is a proinflammatory cytokine that plays an important role in the T-helper type 2 cell pathway. The effects of IL-25 are mediated by its specific receptor, IL-17RB. Previous studies have defined an IL-17RB+ granulocyte population known as type 2 myeloid (T2M) cells that express T-helper type 2 cell cytokines. The correlation of IL-17RB+ granulocytes, T2M cells, and asthma parameters is unknown. OBJECTIVE: To investigate the relation of IL-17RB+ granulocytes (and its subset, T2M cells) in patients with asthma with clinical parameters including spirometric values and the Asthma Control Test (ACT). METHODS: Peripheral blood from subjects with asthma and healthy controls was collected and analyzed by flow cytometry. Granulocytes were gated for IL-17RB+, T2M (CD11b+CD16+CD177+IL-17RB+), and eosinophil (CD16-) populations. Spirometry testing was performed on subjects with asthma. ACT scores and medical histories were collected by questionnaire and chart review. Correlations of IL-17RB+ cells and T2M cells with spirometry and ACT score were analyzed. RESULTS: Percentages of IL-17RB+ granulocytes and T2M cells were larger in subjects with asthma than in controls. Furthermore, percentages of the 2 cell populations were negatively correlated with degree of airway obstruction as measured by the ratio of percentage-predicted forced expiratory volume in 1 second to force vital capacity (r = -0.17, P = .043 for IL-17RB+ granulocytes; r = -0.32, P = .03 for T2M cells). There was no correlation with ACT score. The percentage of eosinophils was increased in subjects with asthma. However, IL-17RB+ eosinophil percentages were similar between subjects with asthma and controls and did not correlate with any clinical parameter. CONCLUSION: IL-17RB+ granulocytes and T2M cells from peripheral blood were increased in subjects with asthma, and the 2 cell types correlated with degree of airflow obstruction.

IPS-1 signaling has a nonredundant role in mediating antiviral responses and the clearance of respiratory syncytial virus.

The cytosolic RNA helicases melanoma differentiation-associated gene 5 and retinoic acid-inducible gene-I and their adaptor IFN-ß promoter stimulator (IPS-1) have been implicated in the recognition of viral RNA and the production of type I IFN. Complementing the endosomal TLR, melanoma differentiation-associated gene 5, and retinoic acid-inducible gene-I provides alternative mechanisms for viral detection in cells with reduced phagocytosis or autophagy. The infection route of respiratory syncytial virus (RSV)-via fusion of virus particles with the cell membrane-points to IPS-1 signaling as the pathway of choice for downstream antiviral responses. In the current study, viral clearance and inflammation resolution were indeed strongly affected by the absence of an initial IPS-1-mediated IFN-ß response. Despite the blunted inflammatory response in IPS-1-deficient alveolar epithelial cells, pulmonary macrophages, and CD11b(+) dendritic cells (DC), the lungs of RSV-infected IPS-1-knockout mice showed augmented recruitment of inflammatory neutrophils, monocytes, and DC. Interestingly, pulmonary CD103(+) DC could functionally compensate for IPS-1 deficiency with the upregulation of certain inflammatory cytokines and chemokines, possibly via TLR3 and TLR7 signaling. The increased inflammation and reduced viral clearance in IPS-1-knockout mice was accompanied by increased T cell activation and IFN-γ production. Experiments with bone marrow chimeras indicated that RSV-induced lung pathology was most severe when IPS-1 expression was lacking in both immune and nonimmune cell populations. Similarly, viral clearance was rescued upon restored IPS-1 signaling in either the nonimmune or the immune compartment. These data support a nonredundant function for IPS-1 in controlling RSV-induced inflammation and viral replication.

The post sepsis-induced expansion and enhanced function of regulatory T cells create an environment to potentiate tumor growth.

One of the more insidious outcomes of patients who survive severe sepsis is profound immunosuppression. In this study, we addressed the hypothesis that post septic immune defects were due, in part, to the presence and/or expansion of regulatory T cells (Tregs). After recovery from severe sepsis, mice exhibited significantly higher numbers of Tregs, which exerted greater in vitro suppressive activity compared with controls. The expansion of Tregs was not limited to CD25(+) cells, because Foxp3 expression was also detected in CD25(-) cells from post septic mice. This latter group exhibited a significant increase of chromatin remodeling at the Foxp3 promoter, because a marked increase in acetylation at H3K9 was associated with an increase in Foxp3 transcription. Post septic splenic dendritic cells promoted Treg conversion in vitro. Using a solid tumor model to explore the function of Tregs in an in vivo setting, we found post septic mice showed an increase in tumor growth compared with sham-treated mice with a syngeneic tumor model. This observation could mechanistically be related to the ability of post septic Tregs to impair the antitumor response mediated by CD8(+) T cells. Together, these data show that the post septic immune system obstructs tumor immunosurveillance, in part, by augmented Treg expansion and function.

Inefficient lymph node sensitization during respiratory viral infection promotes IL-17-mediated lung pathology.

Development of bronchus-associated lymphoid tissue has been suggested to enhance local antiviral immune responses; however, ectopic lymph node formation often corresponds to chronic inflammatory diseases. These studies investigated the role of ectopic pulmonary lymph nodes upon respiratory syncytial virus (RSV) infection using CCR7-deficient mice, which develop bronchus-associated lymphoid tissue early in life. CCR7(-/-) mice exhibited impaired secondary lymph node formation, enhanced effector T cell responses and pathogenic mucus production in the lung after RSV infection. IL-17 production from CD4 T cells in CCR7(-/-) mice was most remarkably enhanced. Wild-type animals reconstituted with CCR7(-/-) bone marrow recapitulated the pathogenic lung phenotype in CCR7(-/-) mice, whereas CCR7(-/-) animals reconstituted with wild-type bone marrow had normal lymph node development, diminished IL-17 production and reduced lung pathology. Mixed bone marrow chimeras revealed an alteration of immune responses only in CCR7(-/-) T cells, suggesting that impaired trafficking promotes local effector cell generation. Lymphotoxin-α-deficient mice infected with RSV were used to further examine locally induced immune responses and demonstrated increased mucus production and amplified cytokine responses in the lung, especially IL-17. Neutralization of IL-17 in CCR7(-/-) or in lymphotoxin-α-deficient animals specifically inhibited mucus hypersecretion and reduced IL-13. Thus, immune cell trafficking to secondary lymph nodes is necessary for appropriate cytokine responses to RSV as well as modulation of the local environment.

The innate immune response to uropathogenic Escherichia coli involves IL-17A in a murine model of urinary tract infection.

Uropathogenic Escherichia coli is the causative agent for >80% of uncomplicated urinary tract infections (UTIs). Uropathogenic E. coli strains express a number of virulence and fitness factors that allow successful colonization of the mammalian bladder. To combat this, the host has distinct mechanisms to prevent adherence to the bladder wall and to detect and kill uropathogenic E. coli in the event of colonization. In this study, we investigated the role of IL-17A, an innate-adaptive immunomodulatory cytokine, during UTI using a murine model. Splenocytes isolated from mice infected by the transurethral route robustly expressed IL-17A in response to in vitro stimulation with uropathogenic E. coli Ags. Transcript expression of IL-17A in the bladders of infected mice correlated with a role in the innate immune response to UTI, and gammadelta cells seem to be a key source of IL-17A production. Although IL-17A seems to be dispensable for the generation of a protective response to uropathogenic E. coli, its importance in innate immunity is demonstrated by a defect in acute clearance of uropathogenic E. coli in IL-17A(-/-) mice. This clearance defect is likely a result of deficient cytokine and chemokine transcripts and impaired macrophage and neutrophil influx during infection. These results show that IL-17A is a key mediator for the innate immune response to UTIs.

3D In Vitro Platform for Cell and Explant Culture in Liquid-like Solids.

Existing 3D cell models and technologies have offered tools to elevate cell culture to a more physiologically relevant dimension. One mechanism to maintain cells cultured in 3D is by means of perfusion. However, existing perfusion technologies for cell culture require complex electronic components, intricate tubing networks, or specific laboratory protocols for each application. We have developed a cell culture platform that simply employs a pump-free suction device to enable controlled perfusion of cell culture media through a bed of granular microgels and removal of cell-secreted metabolic waste. We demonstrated the versatile application of the platform by culturing single cells and keeping tissue microexplants viable for an extended period. The human cardiomyocyte AC16 cell line cultured in our platform revealed rapid cellular spheroid formation after 48 h and ~90% viability by day 7. Notably, we were able to culture gut microexplants for more than 2 weeks as demonstrated by immunofluorescent viability assay and prolonged contractility.

CCL20/CCR6 blockade enhances immunity to RSV by impairing recruitment of DC.

Chemokines are important mediators of the immune response to pathogens, but can also promote chronic inflammatory states. Chemokine receptor 6 (CCR6) is found on immature DC and effector/memory T cells, and binds a single ligand, CCL20, with high affinity. Here, we investigated the role of CCL20 and CCR6 in a pulmonary viral infection caused by RSV, a ubiquitous virus that can cause severe pulmonary complications. Neutralization of CCL20 during RSV infection significantly reduced lung pathology and favored a Th1 effector response. CCR6-deficient animals recapitulated this phenotype, and additionally showed enhanced viral clearance when compared with WT mice. No differences were observed in migration of T cells to the lungs of CCR6(-/-) animals; however, a significant reduction was observed in numbers of conventional DC (cDC), but not plasmacytoid DC, in CCR6(-/-) mice. A pathogenic phenotype could be reconstituted in CCR6(-/-) mice by supplying cDC into the airway, indicating that mere number of cDC dictates the adverse response. Our data suggest that blockade of the CCL20/CCR6 pathway provides an environment whereby the attenuated recruitment of cDC alters the balance of innate immune cells and mediates the efficient antiviral response to RSV.

Identification of key processes that control tumor necrosis factor availability in a tuberculosis granuloma.

Tuberculosis (TB) granulomas are organized collections of immune cells comprised of macrophages, lymphocytes and other cells that form in the lung as a result of immune response to Mycobacterium tuberculosis (Mtb) infection. Formation and maintenance of granulomas are essential for control of Mtb infection and are regulated in part by a pro-inflammatory cytokine, tumor necrosis factor-alpha (TNF). To characterize mechanisms that control TNF availability within a TB granuloma, we developed a multi-scale two compartment partial differential equation model that describes a granuloma as a collection of immune cells forming concentric layers and includes TNF/TNF receptor binding and trafficking processes. We used the results of sensitivity analysis as a tool to identify experiments to measure critical model parameters in an artificial experimental model of a TB granuloma induced in the lungs of mice following injection of mycobacterial antigen-coated beads. Using our model, we then demonstrated that the organization of immune cells within a TB granuloma as well as TNF/TNF receptor binding and intracellular trafficking are two important factors that control TNF availability and may spatially coordinate TNF-induced immunological functions within a granuloma. Further, we showed that the neutralization power of TNF-neutralizing drugs depends on their TNF binding characteristics, including TNF binding kinetics, ability to bind to membrane-bound TNF and TNF binding stoichiometry. To further elucidate the role of TNF in the process of granuloma development, our modeling and experimental findings on TNF-associated molecular scale aspects of the granuloma can be incorporated into larger scale models describing the immune response to TB infection. Ultimately, these modeling and experimental results can help identify new strategies for TB disease control/therapy.

Regulation of T cell activation by Notch ligand, DLL4, promotes IL-17 production and Rorc activation.

The activation and differentiation of T cells are dependent upon numerous initiating events that are influenced by the immune environment, nature of the Ag, as well as the activation state of APCs. In the present studies we have investigated the role of a specific notch ligand, delta-like 4 (Dll4). In particular, our data have indicated that Dll4 is inducible by pathogen-associated signals through TLR activation on dendritic cells but not early response inflammatory cytokines, IL-1 and IL-18 that also activate cells via MyD88 adapter pathway. Our observations from in vitro cultures confirmed earlier reports demonstrating that Dll4 inhibits Th2 cytokine production. Furthermore, Dll4 influences the generation of IL-17-producing T cells in the presence of additional skewing cytokines, IL-6 and TGF-beta. In the absence of notch signals, IL-17 production was significantly inhibited even under specific skewing conditions. These studies further demonstrate that Dll4 up-regulates Rorc expression in T cells and that both Rorc and Il17 gene promoters are direct transcriptional notch targets that further enhance the differentiation of Th17 cell populations. Thus, facilitation of efficient T cell differentiation may depend upon the activation of T cells via specific notch ligand stimulation.