Mastitis and milk quality affect every dairy farmer across the globe. Sand bedded freestalls are the industry standard for cow comfort, welfare, and the control of environmental mastitis. Compost bedded packs may be a viable alternative to the sand bedded freestall. Compost bedded packs are maintained at a consistent level of moisture, nutrients, and aeration to favor compost microorganisms. Greater bacteria counts in bedding have traditionally been associated with increased mastitis rates and mastitis pathogens can be found in the pack and on the teats of cattle housed in even well managed compost bedded pack barns. In spite of this, herd SCC often remains low in well managed herds. The relationship between stress and comfort in the housing environment was a primary focus of this research. Cows housed in environments with low stress and high comfort may be better able to defend themselves against pathogens. Establishing changes in immune function in response to housing environment would improve milk quality by contributing to the knowledge of how mastitis-causing pathogens are contracted. An additional goal of this research was to determine the effect of compost bedded pack barns on thermoduric bacteria populations. Due to the increased temperatures associated with composting, thermoduric bacteria capable of surviving pasteurization are of potential concern in compost bedded packs. This research will investigate potential differences in thermoduric bacteria counts between compost bedded packs and sand bedded freestalls.

Measures of temperament have been shown to influence physiological responses. Exit velocity (EV) has been identified as an objective, robust measure of temperament that can be used to predict subsequent performance of cattle. Additionally, previous studies from our lab indicate this measure of temperament may be related to production of interferon-γ (IFN-γ), a cytokine associated with cell-mediated immunity (CMI). Whereas research has investigated effects of EV upon immune responses, the overall goal of these studies was to examine this relationship under a variety of scenarios including human handling, transportation, and exposure to endophyte-infected tall fescue (E+) for determination of its ability to influence CMI in cattle. In each of 5 experiments, calves were classified as either high or low EV animals, based upon measurements obtained prior to initiation of experimental periods. The hypothesis for these studies was that calves with high exit velocities would have lower systemic immune responses to applied treatments. Two experiments were designed to examine the relationship between exit velocity and lymphocyte IFN-γ production during and following a period of exposure to E+ seed and increased temperature humidity index conditions. Preliminary measures of this cytokine indicated a positive relationship with EV. During application of heat and E+ treatment application, no differences in IFN-γ production were detected between EV or endophyte treatment groups. However, in both experiments, after temperatures were returned to thermoneutral and E+ heifers were placed on the endophyte-free treatment, the positive relationship between exit velocity and total lymphocyte production of IFN-γ observed in baseline samples was reestablished. Similarly, during an experiment examining IFN-γ production by lymphocytes in steers during the 4 weeks following a 10h, 805 km transport study, average lymphocyte production of IFN-γ was higher and lymphocyte proportions producing IFN-γ lower in low EV steers, but total lymphocyte production of this cytokine did not differ between exit velocity treatments. In a grazing and finishing study, cattle were placed on E+ or novel endophyte pastures, with balanced representation of low and high EV treatments within each pasture. During the subsequent finishing period, blood samples for lymphocyte IFN-γ production were collected from a single high EV calf from each pasture group. Neither endophyte nor exit velocity was detected to be related with lymphocyte production of IFN-γ. In an experiment examining changes in cytokine gene expression changes during acclimation to human handling, IFN-γ, Il-6, IL-10, and IL-12 were observed to increase linearly over the experimental period in all calves, irrespective of exit velocity designation. In the same experiment, whole period pro-inflammatory tumor necrosis factor-α expression was higher for high EV calves, but interferon-γ (IFN-γ) was lower in this same treatment group. These studies, cumulatively, indicate EV may be related to systemic production of IFN-γ, but abrupt changes to an animal’s environment may serve to mask this relationship.

Infection with Plasmodium species leads to nearly 400,000 deaths a year despite widespread use of mosquito bed nets, insecticides, and anti-malarial drugs. To date, there is not a licensed vaccine capable of providing complete protection from Plasmodium infection to vaccinees. Whole parasite vaccination of humans and rodents can achieve complete protection in vaccines, but the dose of sporozoites, number of administrations, and production concerns in generating these types of vaccines will likely prevent these approaches from achieving worldwide use. However, the protective immunological responses against Plasmodium parasites engendered by these vaccination approaches can be studied and aid in the development of advanced subunit vaccines against Plasmodium. Using rodent models of malaria to elucidate the features of protective immunity engendered by whole parasite vaccination, it has been repeatedly shown that CD8 T cell responses directed against liver-stage parasite antigens can provide complete protection with some contribution by CD4 T cells and antibody responses depending on the model system studied. However, the quantatitive and qualitative requirements for CD8 T cell immunity against Plasmodium remains largely undefined. To enhance our understanding of how to generate protective immunity against Plasmodium, I have utilized rodent models of malaria to study the superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as chemoprophylaxis sporozoites (CPS) vaccination, compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). RAS vaccination has long been considered the “gold standard” in vaccination due the ability of RAS vaccination to engender complete protection following sporozoite challenge of vaccinated humans and rodents. However, CPS vaccination is arguably a superior vaccination approach since it can achieve protection through less vaccine administrations relative to RAS vaccination, but the immunological basis of this enhanced CPS vaccine-induced immune response was unclear. In my study, I utilized a stringent host/parasite model to find that C57Bl/6 mice administered CPS vaccination with P. yoelii sporozoites elicit substantially higher parasite-specific CD8 T cell responses than RAS vaccination, but CPS-induced CD8 T cells were not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. CPS vaccination resulted in a low grade, transient parasitemia shortly following cessation of chloroquine treatment, which lead to the generation of potent antibody responses to blood-stage parasites; this blood-stage parasite-specific antibody response correlated with sterilizing protection in sporozoite challenged CPS-vaccinated mice. Therefore, my data provide a mechanistic basis for enhanced protective immunity elicited by single-dose CPS vaccination in a rodent model that is independent of CD8 T cells. The other portion of my work examines how CD8 T cell specificity impacts protective capacity against Plasmodium. I show that robust CD8 T cell responses of similar phenotype are mounted following prime-boost immunization against three novel Plasmodium berghei protein-derived epitopes in addition to a previously described protective, immunodominant epitope. I show that only CD8 T cells specific to sporozoite surface-expressed protein-derived epitopes, but not the intracellular protein-derived epitopes, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that antigenic targets must be efficiently presented by infected hepatocytes for CD8 T cells to eliminate liver-stage Plasmodium infection and proteins expressed on the surface of sporozoites may be good target antigens for protective CD8 T cells. Collectively, my work highlights the ability to generate protective CD8 T cell independent and dependent immunity against Plasmodium infections, whether achieved through potent blood-stage-specific antibody responses, or via numerically large monospecific CD8 T cell responses that target parasite antigens that are efficiently presented during liver-stage infection. These studies are relevant in understanding how to efficiency engender protective immunity against Plasmodium, and could aid in the advancement of subunit vaccination approaches that generate immunity through the priming of responses from multiple arms of the immune response, targeting both the liver- and blood-stages of Plasmodium.

Mastitis represents one of the major economical and animal welfare concerns within the dairy industry. Animals affected with this disease can experience a range of clinical symptoms from mild discomfort and swelling of the udder to a severe systemic inflammatory response that could result in the death of the animal. This range of responses is due to differences in pathogen, environment, and inter-animal differences in their innate immune response. A dermal fibroblast model was used to predict the magnitude of an animal's innate immune response towards an intra-mammary S. aureus challenge. Animals whose fibroblasts exhibited a low response phenotype, characterized by lower levels of IL-8 following in vitro immune stimulation, suffered less mammary tissue damage and a less severe reduction in milk quality following the in vivo S. aureus challenge as compared to animals classified as high responders. Furthermore, the heightened inflammatory response of the high responders offered no advantage in bacterial clearance. For a S. aureus infection, the lower response phenotype is preferred. To further explore inter-animal variation in the innate immune response, fibroblast cultures were established and challenged with LPS from two breeds of cattle, Holsteins, a dairy breed and Angus, a beef breed. Cultures from Holstein animals exhibited a higher responding phenotype than cultures from Angus animals. As these two breeds undergo selection for different traits and are reared differently as calves, whole transcriptome analysis (RNA-Seq) and DNA methylation analysis (Methylated CpG Island Recovery Assay; MIRA-Seq) of their fibroblasts was completed to examine the genetic and epigenetic basis for the contrasting responses. RNA-Seq revealed several immune associated genes that were expressed at higher levels in Holstein cultures compared to Angus cultures, including TLR4, IL-8, CCL5, and TNF-α, both basally and following LPS exposure. Although MIRA-Seq analysis revealed 49 regions with differential methylation between the Holstein and Angus cultures, overall, the methylation of the fibroblast genome was similar between these breeds. A combination of genetic and epigenetic factors seems to contribute to the breed-dependent differences observed between Holstein and Angus fibroblasts. Early life exposure to bacterial compounds or inflammatory mediators can have long-term effects on the magnitude of an animal's innate immune response, and may contribute to inter-animal variation in this response. To determine if an early life exposure to LPS would modify the response to a subsequent LPS challenge in dairy animals, neonatal Holstein calves were treated with LPS or saline at 7 days of age and subsequently challenged with LPS 25 days later. Calves that received LPS at 7 days of age had greatly elevated levels of plasma IL-6 and TNF-α compared to calves that received saline, indicating a substantial inflammatory response. However, following the subsequent LPS challenge completed on all calves, there were no differences in plasma IL-6 and TNF-α between the LPS- and saline- treated calves. Alternative exposure strategies in calves may generate the long-term effects observed in other model systems. There is a wide range in the responses observed in the innate immune response of the bovine. Animals with a lower innate immune response effectively clear the infection, but avoid the collateral tissue damage from excessive inflammation. Therefore, it seems that a reduced innate immune response would be more beneficial to the dairy cow.

Periodontal diseases (PD) affect 46% of American adults over age 30. These diseases cause symptoms including bleeding and swelling of the gums, bone resorption, and tooth loss, that affect quality of life and have a high economic burden. Periodontal diseases are caused by an imbalance in the oral microbiome, from a healthy state that contains anti-inflammatory commensals like Streptococcus gordonii and mitis, to a diseased state that has pro-inflammatory anaerobic pathogens including Porphyromonas gingivalis, Prevotella intermedia, Fusobacterium nucleatum, and Tannerella forsythia. The latter initiate disease progression in the oral cavity. However, it’s the host immune response that causes a majority of the symptoms. Ideally, treatment for PD would be approached from both sides to reduce the numbers of pro-inflammatory bacterial cells in the oral cavity but also reduce the host immune response. A novel therapeutic, amixicile, has been created, which specifically targets anaerobes through the pyruvate:ferredoxin oxidoreductase (PFOR) system, the mechanism of energy metabolism found in anaerobic organisms. Our studies show that amixicile inhibits in vitro growth of oral anaerobes in monospecies cultures at concentrations as low as 0.5 µg/mL in broth and 1 µg/mL in biofilms, without affecting the Gram-positive commensal species. In multispecies cultures, amixicile specifically inhibited anaerobes, even in biofilms, with the concentration as low as 5 µg/mL in broth and 10 µg/mL in biofilms. By not affecting the commensal bacteria, we think this treatment could restore a healthy oral microbiome. Aside from the bacterial presence, the host response, particularly the innate immune response is not well understood. Using a Drosophila melanogaster infection model, we elucidated the innate immune response to both mono- and multispecies infections. The 7-Species infection included bacteria mentioned above and Aggregatibacter actinomycetemcomitans in order to replicate in vivo-like disease conditions. We determined that both Drosophila Toll and Imd pathways, which mimic TLR/IL-1 and TNF signaling pathways of mammalian innate immunity respectively, respond to the 7-Species challenge. We also verified virulent bacteria in Drosophila, including P. gingivalis and P. intermedia. Future directions include RNA sequencing to determine the full scope of immune gene expression and using human immune cells to further clarify the response.

The proper regulation of inducible costimulator (ICOS) and its ligand (ICOSL) have been shown to be essential for maintaining immune homeostasis. Loss of either protein results in defective humoral immunity, and overexpression of ICOS results in aberrant antibody production resembling lupus. How ICOSL is regulated in response to ICOS interaction is still unclear. We demonstrate that ADAM10 is the primary physiological sheddase of ICOSL in both mouse and human. Using an in vivo system in which ADAM10 is deleted only on B cells (ADAM10B-/-), elevated levels of ICOSL were seen. This increase is also seen when ADAM10 is deleted from human B cell lines. Identification of the primary sheddase has allowed the characterization of a novel mechanism of ICOS regulation. In wildtype (WT) mice, interaction of ICOSL/ICOS results in ADAM10 induced shedding of ICOSL on B cells and moderate ICOS internalization on T cells. When this shedding is blocked, excessive ICOS internalization occurs. This results in severe defects in T follicular helper (TFH) development and Th2 polarization, seen in a house dust mite exposure model. In addition, enhanced Th1 and Th1 immune responses are seen in experimental allergic encephalomyelitis. Blockade of ICOSL rescues T cell ICOS surface expression and at least partially rescues both TFH numbers and the abnormal antibody production previously reported in these mice. Overall, we propose a novel regulation of the ICOS:ICOSL axis, with ADAM10 playing a direct role in regulating ICOSL as well as indirectly regulating ICOS, thus controlling ICOS:ICOSL-dependent responses. Additionally, we report a specific role for the metalloprotease ADAM10 on B cells in regulating both ICOSL and ICOS in a mouse model of increased humoral immunity using mir146a-/- mice and a model of lymphoproliferative disease using the well characterized lpr model. B6lpr mice lacking ADAM10 on B cells (A10Blpr) have decreased nodal proliferation and T cell accumulation compared to control B6lpr mice. Additionally, A10Blpr mice have a drastic reduction in autoimmune anti-dsDNA antibody production. In line with this, we found a significant reduction in follicular helper T cells (TFH) and germinal center (GC) B cells in these mice. We also show that lymphoproliferation in this model is closely tied to elevated ICOS levels and decreased ICOSL levels. Overall, our data not only shows a role of B cell ADAM10 in controlling autoimmunity, but also increases our understanding of the regulation of ICOS and ICOSL in the context of autoimmunity. Additionally, we found that ADAM17 is important for marginal zone (MZ) B cell development as well as responses to T-independent type 2 (TI2) immunizations. Mice which lack ADAM17 on B cells (A17B) have decreased MZ B cell numbers but have increased levels of antigen specific antibodies in response to TI2 Immunizations. ADAM17 also regulates the level of several surface molecules on plasma cells and MZ B cells necessary for their function and survival. We also show a role for ADAM17 in ILC2 responsiveness to IL-33. In vivo, mice that lack ADAM17 specifically on ILC2s (ADAM17ILC2-/-) exhibit decreased ILC2 expansion in response to intranasal IL-33 as well as Nippostrongylus brasiliensis (Nb) infection. However, ADAM17ILC2-/- mice have normal ILC2 numbers in a naïve state, suggesting this defect in ILC2 function is limited to cell activation. In vitro, ADAM17 inhibited ILC2s have an increased level of apoptosis and less IL-13 production in response to IL-33 compared to vehicle treated ILC2s. The defect in cytokine production following ADAM17 inhibition is not observed in response to IL-25 stimulation, suggesting this defect is limited to IL-33 stimulation Mechanistically, ADAM17 inhibition in ILC2s specifically causes a defect in IL-33 mediated ERK activation, potentially explaining the defective survival and IL-13 production following ADAM17 inhibition in these cells. Additionally, ADAM17 regulates the level of surface IL1R2 which may affect IL-33 signaling in ILC2s.

Pensando nas falhas de transferência de imunidade passiva e nos prejuízos gerados para a equideocultura, este projeto visou avaliar a suplementação com levedura viva, Saccharomyces cerevisiae vivas na dieta de éguas no terço final da gestação sobre a transferência de imunidade passiva, através da avaliação do colostro, concentração de imunoglobulina, perfil celular e composição e desenvolvimento do sistema imune dos potros. O experimento foi conduzido nas dependências do Laboratório de Pesquisa em Saúde Digestiva e Desempenho de Equinos (LabEqui), FMVZ/USP no Campus Fernando Costa em Pirassununga/SP. Foram utilizadas dezesseis éguas prenhes sem raça definida, com idade média de 90±7 meses, mantidas em piquetes sem acesso a gramíneas durante todo o período experimental. A dieta foi formulada para atender a exigência da categoria de acordo com NRC 2007, foi composta de feno de gramínea e concentrado, água e sal mineral ad libitum. As mesmas foram divididas em dois grupos de oito animais cada, 1) grupo controle: feno de Tifton 85 e concentrado comercial sem suplementação, 2) grupo suplementado: feno de Tifton 85 e ração comercial com suplementação de Actisaf HR Plus Sc 47® - 10 g/animal/dia. O delineamento foi inteiramente casualizado (DIC), com medidas repetidas no tempo. Foi observado efeito para células mononucleares no colostro no grupo suplementado (P=0,05) e para células polinucleares para grupo controle (P=0,05). Pode-se concluir que suplementação com levedura viva Saccharomyces cerevisae na dieta de éguas no terço final da gestação, melhora a qualidade do colostro no que se refere ao perfil celular porém não melhora a transferência de imunidade passiva dos potros. / Considering the failures of passive immunity transfer and losses generated for equideoculture, this project aimed to evaluate the live yeast supplement Saccharomyces cerevisiae in the diet of mares in the final third of gestation on the transference of passive immunity through the evaluation of colostrum , immunoglobulin concentration, cellular profile and composition and development of the immune system of foals. The experiment was conducted at the Laboratory of Research on Digestive Health and Performance of Equines (LabEqui), FMVZ / USP at the Fernando Costa Campus in Pirassununga / SP. Sixteen pregnant mares were used, with mean age of 90 ± 7 months, kept in pickets without access to grasses throughout the experimental period. The diet was formulated to meet the requirement of the category according to NRC 2007, was composed of grass hay and concentrate, water and mineral salt ad libitum. They were divided in two groups of eight animals each, 1) control group: Tifton 85 hay and commercial concentrate without supplementation, 2) supplemented group: Tifton 85 hay and commercial feed with Actisaf HR Plus Sc 47®-10 supplementation g / animal / day. A completely randomized (DIC) design, with measures repeated over time was considered in the model. There was observed a statistical effect of supplementation (P = 0.05) over mononuclear cells in colostrum and for polynuclear cells (P = 0.05). It can be concluded that supplementation with live yeast Saccharomyces cerevisae in the diet of mares in the final third of gestation may improve quality of colostrum with respect to the cellular profile, but does not improve the transference of passive immunity of the colts.

The increasing threat of epidemic and pandemic influenza underscore the need to better-understand the immune response to influenza virus infections and to better understand the factors that contribute to the clearance of virus without complications of immunopathology. A hallmark of the adaptive immune response to primary influenza virus infections is the induction of influenza-specific CD8+ T cell responses. These T cells target and kill influenza-infected epithelial cells in the airway, thereby clearing the virus and allowing recovery of the infected host. Recent reports demonstrated that CD8+ T cells express TNF-related apoptosis-inducing ligand (TRAIL) after influenza virus infection. While roles for perforin/granzyme and Fas:FasL interactions in clearing influenza virus infections had been established, little was known about the role of TRAIL in the CD8+ T cell responses to influenza virus infection. We hypothesized that influenza-specific CD8+ T cells would express TRAIL after influenza infection and could utilize TRAIL to induce the apoptosis of virally-infected cells. We discovered that CD8+ T cells do express TRAIL after influenza infection, and that this expression occurs in an influenza-specific fashion. Further, we demonstrated that these influenza-specific CD8+ T cells utilize this TRAIL to kill virally infected cells and protect the host from death, while T cells lacking TRAIL were unable to kill targets as efficiently and provided reduced protection. These data supported our hypothesis that CD8+ T cells utilize TRAIL to kill infected cells. Unexpectedly, when we increased the initial viral inoculum, the pulmonary cytotoxicity of T cells in TRAIL-/- mice was increased compared to those in TRAIL+/+ mice. Investigation of this phenomenon revealed that changes in cytotoxicity correlated not with changes in effector molecule expression on the T cells, but with increased recruitment of T cells to the lung. T cell recruitment to the lungs of TRAIL-/- mice was dependent on CCR5 and CXCR3, and likely the result of aberrant expression of MIG and MIP-1α in the lungs. Together, these data suggest that TRAIL expression contributes not only to T cell cytotoxicity, but also to the regulation of chemokine expression and associated cell recruitment after influenza virus infections. To confirm the relevance of our animal model to the study of human disease, we examined the potential role for TRAIL in the human immune response to infection. We determined that in vitro influenza infection stimulates upregulation of functional TRAIL on the surface of CD3+, CD14+, CD19+, and CD56+ PBMC populations. This expression was not caused by infection of the cells, but by interferon produced as a result of the infection. Infected (TRAIL-expressing) PBMCs killed influenza-infected lung epithelial cells, revealing that influenza infection sensitizes epithelial cells to TRAIL-induced apoptosis. Surprisingly, blocking TRAIL signaling, but not FasL signaling, was able to abrogate this killing of infected epithelial cells. Together, these data support a role for TRAIL in the human immune response to influenza virus infections. Considered as a whole, the data from these studies suggest an additional, previously-unappreciated mechanism by which CD8+ T cells can kill virally infected cells, TRAIL. They also suggest additional, previously-unappreciated roles for TRAIL in immune responses: in helping clear virally infected cells after infections and in helping control cytokine/chemokine expression, and thus the immune response, after virus infection.

Neutrophils (PMN) and group IIA phospholipase A2 (gIIA PLA2) are components of the innate immune system mobilized to sites of invasion by microorganisms such as Staphylococcus aureus. Although accumulating coincidentally in vivo, the in vitro anti-staphylococcal activities of PMN and gIIA PLA2 have thus far been separately studied. The goal of this thesis was to study the collaborative activity of PMN and gIIA PLA2 against S. aureus. We have identified and characterized the collaboration of PMN and gIIA PLA2 against S. aureus ingested by PMN. PMN induced conversion of bacterial phosphatidylglycerol into cardiolipin, but were unable to degrade S. aureus phospholipids without gIIA PLA2. PMN reduced by 10-fold the concentration of gIIA PLA2 needed to digest bacterial phospholipids alone. In addition to increased phospholipid degradation, collaboration of PMN and gIIA PLA2 caused greater bacterial killing and greater loss of bacterial green fluorescent protein fluorescence. The collaboration of PMN and gIIA PLA2 against S. aureus is dependent on catalytic activity and is specific to gIIA PLA2 as related secretory PLA2, groups IB, V, and X, show little or no phospholipid degradation of S. aureus either alone or in the presence of PMN. Synergy of PMN and gIIA PLA2 requires a functional NADPH oxidase and phagocytosis. Although addition of gIIA PLA2 after phagocytosis causes some bacterial phospholipid degradation, the greatest effect is observed when gIIA PLA2 is added before phagocytosis. An extracellular source of H2O2 can partially restore antibacterial activities to NADPH oxidase deficient PMN including the ability to collaborate with gIIA PLA2, supporting a role for reactive oxygen species in NADPH oxidase dependent antimicrobial functions of PMN. In contrast, iberiotoxin, an inhibitor of BK potassium channels had no effect of PMN antibacterial activities. Although H2O2 partially restored antibacterial activity to NADPH oxidase deficient PMN, extracellular H2O2 was not sufficient to increase S. aureus to gIIA PLA2 activity. In summary, PMN and gIIA PLA2 collaborate against S. aureus. These findings revealed collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.

Tularemia is a potentially fatally illness caused by the facultative intracellular Gram-negative bacterium Francisella tularensis. Virulent strains of F. tularensis can cause a fatal disease after inhalation of a few as ten organisms. Due to the highly pathogenic features of Francisella, it has been designated as a Tier 1 select agent, meaning that its possession and handling is highly restricted. Macrophages are phagocytes that play a central role in the innate immune response to infection that can be used by certain pathogens, including Francisella, as a niche for bacterial replication and dissemination during infection. After infection of macrophages Francisella escapes from the phagosome and replicates in the cytosol, however the bacterial factors required for these aspects of virulence are incompletely defined. Here we describe the isolation and characterization of F. tularensis subspecies tularensis strain Schu S4 mutants in iglI, iglJ, and pdpC, three genes located in the Francisella Pathogenicity Island. Our data demonstrate that these mutants were unable to replicate in macrophages due to a defect in phagosome escape. However, a small percentage of pdpC mutants were able to reach the cytosol and replicate moderately. Both iglJ and pdpC mutants were highly attenuated for virulence in a mouse intranasal infection model, however pdpC but not iglJ mutants, were able to disseminate from the lung before eventual clearance. These data demonstrated that the FPI genes tested were essential for F. tularensis Schu S4 virulence, but suggest that they may have different functions due to the unique phenotype observed for pdpC mutants. Our studies also characterized the role of F. tularensis O-antigen and capsule to facilitate interactions with components of the serum complement system; demonstrating that the O-antigen is required for binding of IgM to the bacteria in order to initiate complement opsonization. IgM dependent complement opsonization of both F. tularensis Schu S4 and LVS strains facilitated enhanced phagocytosis of the bacteria by complement receptors 3 and 4 of human macrophages. In addition, we examined the mechanisms of macrophage cytotoxicity and proinflammatory cytokine secretion that was induced after infection with a Schu S4 LPS O-antigen and capsule mutant. The response to the mutant was dependent on phagosome escapes, suggesting a cytosolic pattern recognition receptor was involved in recognition of the bacteria. We found that the cytotoxic and proinflammatory responses had both similar and distinct requirements between human and murine macrophages. Infection with the O-antigen mutant induced robust proinflammatory cytokine secretion that was dependent on caspase-1, cathepsin B, and ASC while cytotoxicity was partially dependent on these molecules. Importantly, we demonstrated that wild-type Schu S4 predominately activated apoptotic caspases, and not inflammatory caspases, during infection and had a blunted cytotoxic response. This was in contrast to the robust cytotoxicity and activation of inflammatory caspases after infection with the non-virulent strain LVS. Together, these studies demonstrated that the Schu S4 LPS O-antigen and capsule are required for evasion of macrophage cytosolic host defense mechanisms.