Diabetes and obesity are associated with inflammation and activation of the immune system with infiltration of adipose tissue by macrophages. This is mainly studied in adipose tissue, with limited information to clarify immune-skeletal muscle interactions in these conditions. We show that exposure of L6 rat skeletal muscle cells to saturated fatty acid palmitate results in insulin resistance, activation of inflammatory pathways, upregulation of pro-inflammatory cytokine and chemokine gene expression and secretion. We identified monocyte chemoattractant protein-1 [MCP-1] as the main factor responsible for macrophage attraction, as blocking it reduced macrophage migration to muscle cells. When macrophages are exposed to palmitate, a similar response ensues with production of macrophage chemoattractants and activation of inflammatory pathways and gene expression profiles, and secretion of multiple cytokines. Our work identifies MCP-1 chemokine produced in response to palmitate treatment by both muscle cells and macrophages and provides a potential link in immune-metabolic crosstalk in diabetogenic environment.

Lfc is a Rho specific guanine nucleotide exchange factor (GEF) that is bound and inhibited by the microtubule (MT) cytoskeleton. In epithelial cells, Lfc promotes actomyosin contractility in response to MT depolymerization; however, its role in leukocytes has not been assessed. Through genetic ablation, we generated an Lfc knockout mouse (Lfc-/-) and tested biochemical and cell biological responses to MT depolymerization in bone marrow derived cells. Lfc was necessary for characteristic actomyosin based contractile behaviours of neutrophils and macrophages, in response to MT depolymerization. Gout is a painful arthritic inflammatory disease, caused by buildup of monosodium urate (MSU) crystals in the joints. Colchicine, a MT-depolymerizing agent that is used in prophylaxis and treatment of acute gout flare, blocks neutrophil infiltration to sites of MSU crystal-induced inflammation. We found that Lfc was necessary for the ability of colchicine to inhibit MSU-induced neutrophil infiltration in two in vivo models of gout-like inflammation. Efficient recruitment of leukocytes from the vasculature is a critical step in the immune response to infection. Leukocyte extravasation, which includes rolling, crawling, and diapedesis across the endothelial barrier, is enhanced by fluid shear stress. Through comparison of Lfc+/+ and Lfc-/- mice, we found that Lfc was necessary for in vivo leukocyte crawling and emigration out of the vasculature. Lfc-/- mice also showed defective neutrophil infiltration in response to acute inflammatory insults, and increased mortality in response to polymicrobial infection. In vitro, we found that Lfc was necessary for neutrophil responses to shear stress.

Specialized cells of the innate immune system, such as macrophages, employ lysosomal enzymes, together with cationic peptides and reactive oxygen intermediates, to eliminate invading microorganisms ensnared within phagosomes. The effectiveness of this impressive armamentarium is potentiated by the acid pH generated by the vacuolar-type ATPase (V-ATPase). The determinants of the luminal pH of phagosomes and of the lysosomes they fuse with are not completely understood, but the V-ATPase is known to be electrogenic and net accumulation of protons requires charge compensation. For this reason, counter-ion pathways are thought to serve a central role in the control of acidification. It has generally been assumed that a parallel anion influx accompanies proton pumping to dissipate the voltage that tends to build up. In fact, impaired chloride channel activity in cystic fibrosis has been proposed to underlie the defective phagolysosome acidification and microbial killing reported in lung macrophages. In the first part of this thesis, I devised methods to dialyze the lumen of lysosomes in intact cells, while monitoring lysosomal pH, in order to assess the individual contribution of counter-ions to acidification. Surprisingly, anions were found to be completely dispensable for proton pumping, whereas the presence of permeant cations in the lysosomal lumen was essential. Accordingly, defects in lysosomal anion permeability cannot explain the impaired microbicidal capacity of phagocytes in cystic fibrosis. Even though counter-ion permeation pathways exist, dissipation of the electrical contribution of the V-ATPase may not be complete. If present, a transmembrane potential would alter the rate and extent of proton accumulation in phagosomes and lysosomes. However, no estimates of the voltage across the phagosomes were available. To overcome this deficiency, in the second part of this thesis, I describe a noninvasive procedure to estimate the voltage across the phagosome using fluorescence resonance energy transfer. This novel approach, in combination with organellar pH measurements, demonstrated that proton pumping is not limited by counter-ion permeability.

Le paludisme, une maladie inflammatoire, est caractérisé par une production du facteur de nécrose tumoral (TNF)-α, du facteur d'inhibition de migration des macrophages (MlF) et d'interféron (lFN)-y, qui inhibent les précurseurs érythropoïétiques de la moelle osseuse. Dans ce contexte, le MlF est sécrété durant les infections par Plasmodium, et sa synergie avec l'lFN-y et le TNF-α contribue à inhiber la différenciation érythropoïétiques et la production d'hémoglobine (Hb). Cet travail à démontrer que la neutralisation in vivo du MlF avec un anticorps monoclonal durant l'infection par Plasmodium chabaudi adami DK amène à une diminution du pic de la parasitémie et de façon inattendue à une production accrue de TNF-α en début d'infection et au pic d'infection. La neutralisation du MlF altère la production d'IFN-y et l'IL-10 durant l'infection. Au moment où la parasitémie est faible, une diminution significative de la production d'lFN-y, accompagnée par une chute importante de l'IL-10, est observée dans la rate de souris infectées et traitées avec l'anti-MIF. Par contre, au pic de l'infection, la production de l'IFN-y et l'IL-10 augmente chez les souris traitées avec l'anti-MIF. En plus de ces effets inhibiteurs au pic de l'infection, la neutralisation du MlF entraîne une diminution du pourcentage de réticulocytes en circulation en début d'infection et cet effet est accompagné par une légère diminution des érythrocytes basophiliques (EryA) dans la rate. La concentration d'hémoglobine sanguine était plus élevée chez les souris traitées avec l'anticorps anti-MIF, et ce, au pic de l'infection, ce qui suggère que la neutralisation du MlF, par son effet sur la parasitémie, diminue la destruction des érythrocytes par le parasite. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Paludisme, Hémoglobine, Cytokine inflammatoire, MlF, Parasitémie.

Les infections par Plasmodium induisent des changements importants sur la réponse effectrice et la régulation des cellules B. Ces effets se caractérisent par une suppression de la production d'anticorps contre des antigènes hétérologues et par une activation polyclonale non spécifique des cellules B. Nous avons centré notre attention sur le facteur inhibiteur de la migration des macrophages (MlF), une cytokine secrétée de façon constitutive chez plusieurs types cellulaires et intensifiée durant l'infection par Plasmodium. Le MlF inhibe l'apoptose des cellules B «in vitro» et son rôle dans les altérations des cellules B durant la malaria n'a pas encore été évalué. En utilisant des souris BALB/c infectées avec P. chabaudi adami DK avant et suite à un régime d'immunisation avec de la γ-globuline humaine, nous avons observé que l'infection après l'immunisation réduit significativement les titres d'lgG totaux et IgGI anti-γ-globuline humaine. Le possible rôle du MlF a été vérifié en neutralisant des souris BALB/c (immunisées avec de la γ-globuline humaine) avec anti-MIF. Les résultats démontrent une augmentation significative d'lFN-γ; une diminution de cellules B anexine +; mais aucun effet significatif sur la production d'anticorps. Étant donné que le MlF est présent pendants les infections nous avons neutralisé le MlF avant et après l'infection par P. chabaudi DK. L'inhibition dans les titres d'anticorps spécifiques à la γ-globuline induit par l'infection était comparable chez des souris traitées avec l'anti-MlF, ce qui suggère que le MlF n'y participe pas. Malgré les effets suggérés «in vitro» sur des cellules B, nos études ont démontré une diminution de cellules B, PI+ chez des souris infectées et neutralisées. En conclusion, nous pouvons dire que le MlF ne semble pas participer dans l'activation polyclonale non spécifique des cellules B durant la malaria et nous suggérons d'évaluer le temps de vie des anticorps en évaluant l'expression du récepteur néonatale Fc (FcRn) qu'y est impliqué à la régulation, le rôle des cellules T régulatrices ainsi que la modulation de l'expression du complexe CD74-CD44 durant les infections par Plasmodium. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Malaria, Cellules B, Réponse d'anticorps, MlF, Activation polyclonale.

Although mechanical ventilator is a life-saving intervention, longer ventilation time and excessive tidal volume contribute to lung injury and increased incidence of infection which is associated with higher mortality. IL-6, a pleiotropic cytokine, participates in both pro- and anti-inflammatory responses. Till now, opinions of the role of IL-6 are widely divided. To study the pathogenesis mechanism of ventilator-induced lung injury (VILI), C57BL/6 mice (WT), IL-6 knockout mice (IL6-/-), chimera (IL6-/- ¡÷ WT) and deletion of I£eB kinase in the myeloid (IKK¡µmye) mice were placed on ventilator for 6 hr. WT mice were also given the IL-6-blocking antibody just before ventilation to evaluate the role of IL-6 signaling in VILI. The results revealed that the pulmonary capillary permeability, neutrophil sequestration, macrophage drifting and protein concentration in bronchoalveolar lavage fluid, and the proinflammatory cytokine levels were significantly increased in ventilated WT mice but not in those pretreated with IL-6-blocking antibody as well as IL6-/-, IKK¡µmye, and IL6-/- ¡÷ WT chimera mice, suggesting that NF-£eB¡VIL-6 signaling could induce inflammation which contributes to the VILI. Furthermore, the antibacterial ability of alveolar macrophages was impaired by ventilation that subsequently increased the danger of developing to ventilator-associated pneumonia.

Equine infectious anemia virus (EIAV) causes rapid development of acute disease followed by recurring episodes of fever, thrombocytopenia and viremia, termed chronic EIA. Most infected horses control the virus by immune mechanisms and become inapparent carriers. To further our understanding of the equine immune response to EIAV, a multi-probe ribonuclease protection assay (RPA) was developed to quantitate equine-specific cytokine mRNAs. Eleven template plasmids specific to ten equine cytokine genes and the ?-actin gene were generated, from which radiolabeled anti-sense RNA probes were produced. The RPA simultaneously quantitated mRNA levels of interleukin (IL)-1, IL-1, IL-6, IL-8, IL-10, IL-12 p35, IL-12 p40, interferon (IFN)-, transforming growth factor (TGF)-1 and tumor necrosis factor (TNF)- in equine peripheral blood mononuclear cells and equine monocyte-derived macrophages (EMDM). The assay detected as few as 5105 RNA molecules and displayed coefficients of variation of 0.03-0.08 when normalized to -actin expression. Using this RPA, cytokine expression in EMDM infected with 2 molecularly cloned viruses (EIAV17 and EIAV19) was determined. EIAV17 varies from EIAV19 only in env, rev and LTR and causes fatal disease in Shetland ponies. When added to EMDM cultures, virulent EIAV17 stimulated expression of IL-1, IL-1, IL-6, IL-10 and TNF-. These cytokine mRNAs were significantly elevated by 0.5 to 1 hr post infection (hpi) and returned to basal levels by 12 to 24 hpi, indicating modulation by early event(s), such as receptor binding. In contrast to EIAV17, EIAV19 is avirulent in vivo and failed to induce any of the tested cytokines in EMDM. These data show a direct correlation between the virulence of the EIAV clone and the induction of cytokines. The cytokines stimulated by EIAV17 may contribute to EIA-associated symptoms, enhance viral replication in the host, and regulate the host immune response. To determine whether cytokine induction requires EIAV17 replication, EMDM cultures were exposed to UV-inactivated EIAV17 and cytokine induction was monitored. UV-inactivation did not block cytokine induction by EIAV17, suggesting dispensability of viral replication. Given that EIAV17 induces cytokines in a rapid and replication-independent manner, the activation of cytokine expression is likely mediated by binding of EIAV17 to equine macrophage receptor(s).

The roles of genes responsible for the survival and persistence of Brucella in the host and the relationship between these genes and the disease were investigated via signature-tagged transposon mutagenesis. As much as 8% of the Brucella genome is important for survival of this organism in the host. This is an unusually high number and may help to explain the chronic or persistent nature of Brucella infections. Mutants attenuated in the mouse model were divided into two groups. The early mutants failed to establish infection or colonize the host. The late mutants colonized the host but failed to maintain infection. The vaccine potential of two mutants (virB10 and gcvH) that were unable to sustain infection was compared to that of a vaccine strain, S19. Survival of strain S19 in vivo was up to 12 weeks while virB10 and gcvH mutants were cleared from spleen at 8, and 24 weeks post-inoculation, respectively. Mice were vaccinated with individual mutants and then challenged with virulent S2308 at 8, 16, and 24 weeks postvaccination. As a result, protective immunity correlated with persistence of the mutant strain [gcvH>virB10]. These results suggest that survival is one of several factors that may influence protective immunity making it difficult to compare strains. For example, examination of host immune response revealed a similar pattern of host immune function (TH1 over TH2) in all mice except those vaccinated with virB10 mutant. Since gcvH mutant provided the best immunity, experiments were designed to explore its contribution of persistence to protection. In an effort to reduce non-specific activation induced by prolonged survival of gcvH mutant, protection was monitored after different periods of vaccination exposure followed with doxycycline treatment. In these studies, persistence of gcvH mutant enhanced protection against challenge. Overall, defined mutations in genes affecting survival may render mutants as vaccine candidates capable of stimulating protective immunity equal to or better than fortuitously isolated attenuated strains. Future studies should focus on characterization of these and other genes responsible for the persistence of Brucella to improve the safety and efficacy of live vaccines.

Thesis (Ph. D.)--Ohio State University, 2009. / Title from first page of PDF file. Includes vita. Includes bibliographical references (p. 119-139).

Skeletal muscle regeneration involves a tightly regulated coordination of cellular and signaling events to remodel and repair the site of injury. When this coordination is perturbed, the regenerative process is impaired. The expression of insulin-like growth factor-I (IGF-I) is robust in the typical muscle regenerative program, promoting cell survival and increasing myoblast activity. In this project, we found that severely depressed IGF-I expression and intracellular signaling in aged skeletal muscle coincided with impaired regeneration from ischemia/reperfusion (I/R). To hasten muscle regeneration, we developed the PEGylated fibrin gel (PEG-Fib) system as a means to intramuscularly deliver IGF-I in a controlled manner to injured muscle. This strategy resulted in greatly improved muscle function and histological assessment following 14 days of reperfusion, which are likely mediated by improved myofiber survival. Recent evidence suggests macrophages (MPs) are responsible for the upregulation of IGF-I following injury, therefore we developed a rapid, reproducible, and cost-effective model of investigating MP profiles in injured muscle via flow cytometry. Using information gathered from this model, we found that increasing the number of a non-inflammatory MP population improves the recovery of muscle from I/R. These data demonstrate that immunomodulatory therapies have the potential to greatly improve the recovery of skeletal muscle from injury. / text