Transient ablation of regulatory T cells has been shown to be effective at hindering tumor growth and metastasis in murine breast cancer model. Based on our lab’s previous work, we have demonstrated that NK cells and CD8+ cytotoxic T cells were not required for the protective effect of Treg cell ablation. However, we also reported that CD4+ helper T cells and IFN-γ were required for the protective effect of Treg cell ablation. Furthermore, we observed that CD11B+ cells responded to Treg ablation therapy by up-regulating target genes of IFN-γ. Therefore, this study aimed to investigate the connection between the myeloid cell compartment and IFN-γ signaling after regulatory T cell ablation therapy. Through a combination of conditional knockout mouse models, cellular fate mapping experiments, adoptive transfers, and co-injection experiments, we demonstrated that tumor-associated macrophages (TAMs), derived from the bone marrow via CCR2/CCL2 axis, were responsible for the therapeutic effect of regulatory T cell ablation. In addition, we determined that IFN-γ signaling was required for the TAMs to mediate the protective phenotype seen after regulatory T cell ablation. Furthermore, based on our findings, we developed a genetic signature based on TAMs from treated or untreated tumors that had a predictive value for patient survival. Thus, our findings indicated a strong connection between IFN-γ release, classical activation of TAMs, and depletion of regulatory T cells, and taken together, our data could offer potential clinical strategies to mimic regulatory T cell ablation.

Western equine encephalitis virus (WEEV) is an arboviral pathogen naturally found in North America. The primary disease phenotype associated with WEEV infection in susceptible hosts is a relatively long prodromal period followed by viral encephalitis. By contrast, in the current work, experimental inoculation of WEEV into the peritoneum of Syrian golden hamsters produced rapid death within approximately 96 h. It was determined that direct virus killing of lymphoid cells leads to death in WEEV-infected Syrian golden hamsters, and that inflammatory cytokines have the potential to enhance virus-induced lymphoid cell destruction. It was further concluded that WEEV retains its ability to cause encephalitis in Syrian golden hamsters, if hamsters survive the early stages of virus infection or if virus is introduced directly into the CNS. Death in WEEV-infected hamsters is associated with lymphonecrotic lesions in the absence of pathological lesions in the central nervous system (CNS). Few clinical parameters were altered by WEEV infection, with the exception of circulating lymphocyte numbers. Circulating lymphocyte numbers decreased dramatically during WEEV infection, and lymphopenia was identified as a consistent indicator of eventual death. Virus infection also increased serum concentrations of the cytokines interferon and tumor necrosis factor-alpha (TNF-alpha). Hamster peritoneal macrophages exposed to WEEV expressed TNF-alpha in a dose-responsive manner. Macrophage expression of TNF-alpha could be significantly inhibited by treatment of cells with anti-inflammatory agents flunixin meglumine (FM) or dexamethasone (Dex). Anti-inflammatory treatment also protected macrophages from cytotoxicity associated with exposure to WEEV. Treatment of WEEV-infected hamsters with either FM or Dex significantly improved survival compared to placebo-treated controls. WEEV induced cytotoxicity in hamster splenocytes exposed to WEEV in a virus dose-responsive manner. Supernatant from WEEV-exposed macrophages significantly enhanced WEEV killing of splenocytes. Hamsters that survived the early stages of WEEV infection occasionally developed signs of neurological disease and died approximately 6 to 9 d after virus inoculation. These animals had histopathological lesions in the CNS consistent with alphavirus-induced encephalitis. Inoculation of WEEV directly into the CNS caused apparent encephalitic disease. Death following CNS inoculation of WEEV was rapid and concurrent with histopathological lesions in the CNS similar to lesions seen in encephalitic hamsters following peripheral inoculation.

Mastitis, or mammary gland inflammation, causes the greatest loss in profit for dairy producers. Mastitis susceptibility differs among cows due to environmental, physiological, and genetic factors. Prior research identified a genetic marker in a chemokine receptor, CXCR1, associated with mastitis susceptibility and decreased neutrophil migration. Current research seeks to identify reasons behind mastitis susceptibility by validating this model through in vivo challenge with Streptococcus uberis and studying specific mechanisms causing impaired neutrophil migration. Holstein cows with GG (n=19), GC (n=28), and CC (n=20) genotypes at CXCR1+777 were challenged intramammarily with S. uberis strain UT888. After challenge 68% of quarters from GG genotype, 74% from CC genotype and only 47% from GC genotype cows had ≥10 colony forming units/ml S. uberis for at least two sampling time points (P<0.05). However, among infected cows, number of S. uberis, somatic cell count, rectal temperature, milk scores and mammary scores were comparable among genotypes throughout infection. These findings suggest that cows with GC genotypes may be more resistant to S. uberis mastitis, but have similar responses if infected. To better understand the mechanisms associated with disease resistance, migration patterns in neutrophils from cows with different CXCR1+777 genotypes were evaluated. Neutrophils from cows with GG (n=11) and CC (n=11) genotypes were isolated and stimulated with zymosan activated sera (ZAS). Cells were fixed and stained for F-actin and evaluated for F-actin content, distribution, and cell morphology. Neutrophils from CC cows had significantly lower average F-actin polymerization than GG cows v (P=0.05). Directed migration of neutrophils from GG (n=10) and CC (n=10) genotypes was imaged and tracking data was analyzed for individual cells. Cells from GG genotype traveled further on an X axis and had higher X/Y movement towards IL8 compared to CC genotype, meaning they moved more directly towards IL8. Our findings suggest lower F-actin polymerization in combination with lower ability to directly move towards IL8 could impair neutrophil response to infection in cows with a CC genotype and may contribute to increased mastitis susceptibility. Finding what makes certain cows more susceptible to mastitis could lead to strategies aimed at improved prevention and treatment of mastitis.

Bacteriophages affect bacterial evolution, pathogenesis and global nutrient cycling. They are also the most numerous and diverse group of biological entities on the planet [1, 2, 3, 4, 5, 6]. Members of the Lambda phage family share a similar genetic organization and control early gene expression by suppressing transcription, a process known as antitermination. Transcription antitermination in Lambda is mediated by a phage-encoded protein whereas in lambdoid phage HK022, antitermination is mediated by a phage-encoded RNA molecules. Recent results suggest that another bacteriophage called HK639 also appears to use RNA-mediated antitermination. To characterize this newly identified phage we generated site directed mutations and identified where the phage integrates into the chromosome of its bacterial host.

Immunosenescence results in reduced immune response to infections with Streptococcus pneumoniae as well as to pneumococcal polysaccharide vaccines. The antibody response to the capsular polysaccharide (CPS) provides protection against S. pneumoniae infection. CPS immunoresponse is T cell independent and needs the macrophage-derived cytokines such as IL-12, IL-6 and IL-1β to elicit an antibody response. We showed a cytokine dysregulation, i.e. a decrease in IL-12, IL-6 and TNF-α but an increase in IL-10, in the aged (18-24 months old comparable to >65 years in human) compared to young adult mouse (8-12 weeks less than 65 years old) splenic macrophages (SM) or bone marrow derived macrophages (BMDM) activated via TLR4, TLR2 or TLR9 as well as heat killed Streptococcus pneumoniae (HKSP). There is also an age-associated defect in splenic B cells in the production of IgG3 upon stimulation with these ligands. A microarray analysis in SM followed by validation by both qt-RTPCR and western blots indicated that this age-associated defect in aged SM, BMDM and B cells was due to a heightened activity of the PI3K-Akt signaling pathway. We hypothesized that the senescence of immune responses in macrophages and B cells is due to an increase in activity of PI3K/Akt and decrease in the activity of GSK-3, the downstream kinase. Inhibition of the PI3-kinase with either LY294002 or Wortmannin restored the TLR2, 4, 9 and HKSP induced cytokine phenotype of the aged to that of the young adult in both the SM and BMDM and an enhanced IgG3 production in aged mice. We also showed that inhibition of glycogen synthase kinase-3 (GSK-3) the downstream target of the PI3K-Akt signaling pathway with SB216763 in SM, BMDM and B cells resulted in an enhancement in production of IL-10, IL-6 and IL-1β by macrophages and in B cell activation. Treatment of B cells with SB216763 in the presence of ligands for TLR-1/2, 4 or 9 as well as HKSP under in vitro conditions led to enhanced production of IgG3 and IgA, plasma cell formation and a slight increase in the proliferation of the B-cells with no adverse effects on the viability of the cells. Therefore, targeting the PI3K-AKT-GKS-3 signaling pathway could rescue the intrinsic signaling defect in the aged macrophages, increase IL-12 and IL-6, and enhance anti-CPS antibody responses.

Abstract can be viewed in PDF document. / Thesis (Ph.D)-University of KwaZulu-Natal, Durban, 2008.

A paracoccidioidomicose é uma micose sistêmica caracterizada por ser uma doença granulomatosa. As formas benignas da doença são caracterizadas por uma infecção localizada, contendo granulomas compactos com poucos fungos; já nas formas mais graves, ocorre um processo granulomatoso frouxo com focos de necrose e intensa disseminação fúngica. Objetivou-se avaliar o desenvolvimento de lesões granulomatosas em baço, fígado, pulmão e epiplon de camundongos, após infecção pela via intraperitoneal com o isolado de alta virulência, Pb18, em diferentes períodos de infecção após tratamento com fármacos, os quais possuem mecanismo de ação relacionado com a alteração no balanço entre a síntese e degradação dos produtos do colágeno, interferindo diretamente na formação do granuloma A citocina IFN-<font face=\"Symbol\">g, o antibiótico Tetraciclina e as drogas antiinflamatórias Lumiracoxib e Celecoxib. Avaliamos a presença de alguns componentes do granuloma (colágeno, células do infiltrado inflamatório, de citocinas primordiais para sintese/degradação da MEC do granuloma, presença de P. brasiliensis). / Paracoccidioidomycosis (PCM) is a systemic mycosis that is endemic in Latin America, whose causative agent is the thermal dimorphic fungus Paracoccidioides brasiliensis (Pb). PCM is a granulomatous disease, and the formation of granulomas can be understood as a mechanism of the body to block and limit the invasiveness of the fungus or its antigenic components, once unable to lyse them. Bening forms of the disease are characterized by a localized infection, where granulomasa are compact and contain few fungi. More severe forms present loose granulomatous processes with foci of necrosis and severe fungal. Studies in which granulomatous response was developed in resistant (A/J) and susceptible (B10.A) mice to the high virulence isolate Pb18 showed the presence of different patterns of injuries related to the type of extracellular matrix (ECM) components and the different cells types in the area, suggesting a important role of these elements in the formation and constitution of the granuloma and thus the outcome of infection. In our project, we aimed to evaluate the development of granulomatous lesions in the spleen, liver, lung and omentum of mice susceptible to PCM after intraperitoneal infection with Pb18, at different periods of infection (acute and chronic) with or without treatment with drugs. These drugs have mechanisms of action closely related to the change in the balance between synthesis and degradation of collagen Thus, they interfere directly in the granuloma formation and in maintaining the viability of fungi and also with the development of fibrosis. Which is a common and devastating sequelae of numerous infections including the PCM, with the characteristic proliferation of fibroblasts and deposition of ECM. The treatments were chosen based on prior knowledge on their effects on the course of experimental murine PCM. IFN-<font face=\"Symbol\">g was chosen due to its antifibrotic effect, being an activator of macrophages in infection by P. brasiliensis and increasing the fungicidal effect of neutrophils. The antibiotic tetracycline was used because of its inhibitory effect on the synthesis of extracellular matrix, limiting antimicrobial activity and the ability of collagenase to degrade ECM. Finally, the antiinflammatory drugs Celecoxib and Lumiracoxib (inhibitors of the COX-2 enzyme) 11 were used because they cause an increase in the expression of collagen type III and type IV. We analyzed the components of the granuloma (collagen, inflammatory cells, cytokines essential for synthesis / degradation of the ECM of the granuloma, the presence of P. brasiliensis). Among the cytokines analyzed, we studied the importance of TNF-<font face=\"Symbol\">&#945 in the formation of granulomas and regulation of matrix metalloproteinases (MMP) synthesis and function. We analyzed TGF-<font face=\"Symbol\">b because it negatively modulate the secretion of nitric oxide by macrophages and promote the accumulation of ECM and is believed to be the central mediator of the process of fibrosis in several pathologies. IFN-<font face=\"Symbol\">g was studied because of its correlation to the preferential Th1 immune response in diseases and infectious processes of fungal and bacterial infections, and also because it modulates fibroblast function.

Cytosolic DNA sensing plays a key role in autoimmune and autoinflammatory diseases. STING is a cytosolic adaptor protein which upon activation leads to induction of type I interferons and inflammatory cytokines. Recently, gain-of-function mutations in STING have been identified in patients with an autoinflammatory disease called STING-associated vasculopathy with onset in infancy (SAVI). We compared two independent SAVI mutant mouse models and revealed a hierarchy of immune abnormalities which were dependent on SAVI mutation in lymphocytes. We also showed that bone marrow from the V154M mutant mice transfers disease to the wild-type host, whereas the N153S does not, indicating mutation-specific disease outcomes. Collectively, these mutant mice recapitulate disease features seen in SAVI patients and highlight mutation-specific functions of STING. Other autoimmune mouse models such as DNAseII and DNAseIII-deficient mice, that fail to degrade DNA result in activation of the cGAS STING pathway. Deficiency of this pathway in these mouse models ameliorates lethality. By contrast, we previously reported that STING potently suppresses inflammation in a pristane-induced model of autoimmunity. In this model, we show that both cGAS- and STING-deficient mice exhibit exacerbated disease phenotypes compared to controls. We report that STING constrained TLR activation, and thereby limited autoimmune manifestations. Consistent with this premise, cGAS or STING deficient mice that lack a common TLR chaperone UNC93b develop less severe systemic autoimmunity than cGAS or STING deficient mice that are UNC93b sufficient. Overall, this study demonstrates that STING activation constrains systemic autoimmune disease and has important implications for cGAS STING-directed therapies.

In alcoholic hepatitis (AH), high neutrophil counts correlate with inflammation and poor clinical outcomes. Here, we sought to elucidate the neutrophil-mediated pathogenesis of AH. We revealed that in vivo neutrophil extracellular trap (NET) formation was significantly increased in AH patients and that alcohol alone is sufficient to induce NET formation in neutrophils; thereby, neutrophils increase liver damage through increased NET formation. Moreover, we identify that alcohol-induced NET formation is vital to NETosis and that high-density neutrophils (HDNs) become low-density neutrophils (LDNs) after NET formation in response to alcohol. Through transcriptome profile analysis, we found that genes related to neutrophil activation and immune responses are significantly upregulated in AH HDNs but significantly downregulated in AH LDNs compared to HDNs from healthy subjects. These data suggest that AH HDNs and LDNs have opposing phenotypes: HDNs are activated and more prone to release NETs, while LDNs are functionally incompetent. Consequently, the increase in activated HDNs and defective LDNs are likely associated with an increase in liver damage through NET formation and enhanced susceptibility to infection in AH patients, respectively. Therefore, we evaluated the therapeutic benefits of preventing NET formation in HDNs using peptidyl arginine deiminase 4 (PAD4) inhibition and granulocyte colony-stimulating factor (G-CSF) treatment as well as neutrophil depletion in mice. We observed that in vivo neutrophil depletion and G-CSF treatment prevent NET formation in the liver, thereby significantly reducing liver damage in alcohol-fed mice. Our work identifies the neutrophil/NET-mediated mechanisms of AH pathogenesis and provides insights into therapeutic interventions for AH.

TCR signal strength is critical for CD8+ T cell clonal expansion after antigen stimulation. Levels of the transcription factor IRF4 control the magnitude of this process through induction of genes involved in proliferation and glycolytic metabolism. The signaling mechanism connecting graded TCR signaling to the generation of varying amounts of IRF4 is not well understood. Here, using multiple methods to vary TCR signal strength and measure changes in transcriptional activation in single CD8+ T cells, we connect antigen potency to the kinetics of NFAT activation and Irf4 mRNA expression. T cells that transduce weaker TCR signals exhibit a marked delay in Irf4 mRNA induction resulting in decreased overall IRF4 expression in individual cells and increased heterogeneity within the clonal population. The activity of the tyrosine kinase ITK acts as a signaling catalyst that accelerates the rate of the cellular response to TCR stimulation, controlling the time to onset of Irf4 gene transcription. These findings provide insight into the signal transduction pathway accounting for the reduced clonal expansion of low affinity CD8+ T cells following infection. We also describe another context for ITK activity, autoreactive T cell migration. Here, we connect TCR signaling strength to modulation of selectin binding and autoreactive T cell-mediated pathology in an adoptive transfer model system of autoimmune disease. Understanding the signaling mechanisms linking changes in TCR signaling to CD8 T cell function is important in furthering the understanding of vaccine development and T cell adoptive immunotherapy.