Biomedical subjects

Gary B Huffnagle

Publications and source records attributed to Gary B Huffnagle.

At least 19 recordsLinked to original sources

PubMed2006

Cryptococcus neoformans produces authentic prostaglandin E2 without a cyclooxygenase.

Many single-celled eukaryotes produce prostaglandin-like molecules, but these have not been absolutely verified by mass spectrometry. We have isolated, and identified by liquid chromatography-tandem mass spectrometry, authentic prostaglandin E(2) from Cryptococcus neoformans. Cyclooxygenase inhibitors did not inhibit prostaglandin synthesis, and the cryptococcal genome lacks a cyclooxygenase homolog. Thus, novel enzymes must exist.

Blotting, Western↗

PubMed2006

Immunologic homeostasis during infection: coexistence of strong pulmonary cell-mediated immunity to secondary Cryptococcus neoformans infection while the primary infection still persists at low levels in the lungs.

Maintenance of immunity to persistent pathogens is poorly understood. In this study, we used a murine model of persistent pulmonary fungal infection to study the ongoing cell-mediated immune response. CBA/J mice with low-level persistent Cryptococcus neoformans infection had CD4+ T cells of effector memory phenotype present in their lungs. Although unable to eliminate the primary infection to sterility, these mice displayed hallmarks of immunologic memory in response to rechallenge with C. neoformans: 1) the secondary cryptococcal challenge was controlled much more rapidly, 2) the inflammatory response developed and resolved more rapidly, 3) CD4+ T and CD8+ T cell responses were higher in magnitude, and 4) effector cytokine production by T cells was greatly enhanced. Depletion of CD4+ T cells at the time of secondary challenge adversely affected clearance of C. neoformans from the lungs. These results demonstrate that persistent low-level infection with C. neoformans does not impair the cell-mediated response to the fungus. Although they are relatively free of overt disease, these mice can respond with a rapid secondary immune response if the burden of C. neoformans increases. These data support the concept that immunologically healthy individuals can maintain low numbers of cryptococci that can become a nidus for re-activation disease during immunodeficient states such as AIDS.

Animals↗

PubMed2006

Diversity of the T-cell response to pulmonary Cryptococcus neoformans infection.

Cell-mediated immunity plays an important role in immunity to the pathogenic fungus Cryptococcus neoformans. However, the antigen specificity of the T-cell response to C. neoformans remains largely unknown. In this study, we used two approaches to determine the antigen specificity of the T-cell response to C. neoformans. We report here that a diverse T-cell receptor (TCR) Vbeta repertoire was maintained throughout the primary response to pulmonary C. neoformans infection in immunocompetent mice. CD4+ T-cell deficiency resulted in relative expansion of all CD8+ T-cell subsets. During a secondary immune response, preferential usage of a TCR Vbeta subset in CD4+ T cells occurred in single individuals, but the preferences were "private" and not shared between individuals. Both CD4+ and CD8+ T cells from the secondary lymphoid tissues of immunized mice proliferated in response to a variety of C. neoformans antigens, including heat-killed whole C. neoformans, culture filtrate antigen, C. neoformans lysate, and purified cryptococcal mannoprotein. CD4+ and CD8+ T cells from the secondary lymphoid tissues of mice undergoing a primary response to C. neoformans proliferated in response to C. neoformans lysate. In response to stimulation with C. neoformans lysate, lung CD4+ and CD8+ T cells produced the effector cytokines tumor necrosis factor alpha and gamma interferon. These results demonstrate that a diverse T-cell response is generated in response to pulmonary C. neoformans infection.

Animals↗

PubMed2006

Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax.

Several models of anthrax pathogenesis suggest that early in the infectious process Bacillus anthracis endospores germinate and outgrow into vegetative bacilli within phagocytes before being released into the blood. Here, we define the respective contributions of three phospholipases C (PLCs) to the pathogenesis of B. anthracis. Genetic deletions of the PLCs were made in the Sterne 7702 background, resulting in the respective loss of their activities. The PLCs were redundant both in tissue culture and in murine models of anthrax. Deletion of all three PLC genes was required for attenuation of virulence in mice after intratracheal inoculation. This attenuation may be attributed to the inability of the PLC-null strain to grow in association with the macrophage. Complementation of these defects in both models of anthrax was achieved by expression of the PLC genes in trans. The functional redundancy between PLCs in the virulence of B. anthracis implies that their activities are important for anthrax pathogenesis.

Animals↗

PubMed2006

Effect of a CD4-depleting antibody on the development of Cryptococcus neoformans-induced allergic bronchopulmonary mycosis in mice.

Allergic bronchopulmonary mycosis (ABPM) is a hypersensitivity lung disease in which fungal colonization is accompanied by an allergic response to the fungus. Using a mouse model of ABPM caused by Cryptococcus neoformans infection of C57BL/6 mice, the goal of the present studies was to determine the effect of the CD4-depleting monoclonal antibody GK1.5 on the development of the allergic responses seen during active fungal infection. These results would provide insight into the role of CD4(+) T cells in this disease. Our results show that GK1.5 treatment resulted in attenuation of pulmonary inflammation and eosinophilia in these animals. These mice also had reduced T2 cytokine production and no serum immunoglobulin E production. Absence of CD4(+) T cells did not affect recruitment of CD8(+) T cells to the site of infection; however, the numbers of CD19(+) B cells were severely reduced in the lungs of CD4(+) T-cell-depleted animals. We also examined changes in the pulmonary architecture and found that depletion of CD4(+) T cells was associated with a significant reduction in mucus production and goblet cell metaplasia in these mice. Interestingly, attenuation of Th2 responses in CD4(+) T-cell-depleted animals did not increase the fungal load in their lungs. We also compared development of ABPM in young and mature mice and did not find any differences at any of the time points. Overall, our results show that depletion of CD4(+) T cells prevents the development of Th2 responses seen during ABPM.

Age Factors↗

PubMed2006

Distinct compartmentalization of CD4+ T-cell effector function versus proliferative capacity during pulmonary cryptococcosis.

The activation and expansion of T cells and their acquisition of effector function are key steps in the development of the adaptive immune response. Most infections are predominantly outside of the lymphoid tissues, and it is unclear at what point developmentally and anatomically T cells acquire effector function in vivo. In these studies, we compared the activation and polarization of T cells during murine pulmonary Cryptococcus neoformans infection in the secondary lymphoid tissues and at the site of primary infection. Few CD4(+) and CD8(+) T cells expressed an activated phenotype (CD44(hi,) CD25(+), CD69(+), CD62L(lo), CD45RB(lo)) at the sites of clonal expansion (lymph nodes, spleen, and blood). In contrast, a high percentage of T cells expressed activation markers at the site of primary infection, the lungs. Additionally, the polarization of CD4(+) T cells to interferon-gamma-producing effector cells occurred at the site of infection, the lungs. CD4(+) and CD8(+) T cells from secondary lymphoid organs responded to TCR restimulation by proliferating, whereas T cells from the lungs proliferated poorly. This report demonstrates for the first time that T-cell activation and effector function in secondary lymphoid tissues during fungal infection is characteristically different from that at the site of primary infection.

Animals↗

PubMed2005

Innate immunity for biodefense: a strategy whose time has come.

Defense against biothreat agents requires a broad-spectrum approach. Modulation of the innate immune system might fulfill this requirement. Hackett's previous review of innate immune activation as a broad-spectrum biodefense strategy identified several unresolved questions. The current article is a systematic approach to answering those questions with the focused participation of research groups developing this technology. Our team of academic and industry participants reviewed the promising agents and came to the following conclusions. It is feasible to construct a biodefense platform combining synergistic agents that activate the innate immune system against a broad range of pathogens on the basis of conserved microbial components by using a nasal spray for immune activation in the respiratory and gastrointestinal tracts because these are the most likely routes of attack. It might also be possible to include agents that inhibit molecular events leading to septic shock. Innate immune-activating agents designed to activate Toll-like and other receptors will probably provide protection against the biothreat pathogen spectrum for periods ranging from 2 to 14 days for IFNs up to 26 weeks for immunomodulatory oligonucleotides. Initial treatment is proposed on the first index case or biosensor alert. Boost doses would be required. Harmful inflammation is possible, but thus far, only transient fever has been observed. Autoimmune reaction and retroviral activation have not been seen thus far in preclinical and human trials of many of these compounds. Toll-like receptor agonists caused cytokine production in all subjects tested, but genetic polymorphism reduced the response to IFN in African American subjects.

Aminoquinolines↗

PubMed2005

Leptin corrects host defense defects after acute starvation in murine pneumococcal pneumonia.

RATIONALE: Leptin is an adipocyte-derived hormone that declines dramatically during fasting and plays a pivotal role in the neuroendocrine response to starvation. Previously, we employed leptin-deficient (ob/ob) mice to identify an important role for leptin in the host defense against Klebsiella pneumonia. OBJECTIVES: To assess the effects of fasting on the innate immune response against pneumococcal pneumonia and to determine the effects of maintaining circulating leptin levels on host defense in fasted mice. METHODS: C57BL/6 mice were either fed ad libitum or fasted for 48 h and given an intraperitoneal injection of saline or recombinant leptin (1 microg/g of body weight) twice daily for 48 h before bacterial challenge. Mice were challenged with 10(5) cfu of Streptococcus pneumoniae via the intranasal route. MEASUREMENTS AND MAIN RESULTS: Lung homogenate S. pneumoniae burden was nearly 20-fold greater in the fasted as compared with fed mice. The impairment in bacterial clearance observed in fasted animals was associated with reduced bronchoalveolar lavage neutrophil counts and interleukin-6 and macrophage inflammatory protein-2 levels. Alveolar macrophages from fasted animals also exhibited defective phagocytosis and killing of S. pneumoniae and reduced calcium-ionophore-stimulated leukotriene B(4) synthesis in vitro. In contrast, the provision of exogenous leptin to fasted animals restored bacterial clearance, bronchoalveolar lavage levels of neutrophils and cytokines, alveolar macrophage bacterial killing, and leukotriene B(4) synthesis. CONCLUSIONS: These results suggest that reduced leptin levels substantially contribute to the suppression of pulmonary antibacterial host defense during starvation and that administration of this adipokine may be of therapeutic benefit clinically.

Acute Disease↗

PubMed2005

Generation of antifungal effector CD8+ T cells in the absence of CD4+ T cells during Cryptococcus neoformans infection.

Immunity to the opportunistic fungus Cryptococcus neoformans is dependent on cell-mediated immunity. Individuals with defects in cellular immunity, CD4(+) T cells in particular, are susceptible to infection with this pathogen. In host defense against a number of pathogens, CD8(+) T cell responses are dependent upon CD4(+) T cell help. The goal of these studies was to determine whether CD4(+) T cells are required for the generation of antifungal CD8(+) T cell effectors during pulmonary C. neoformans infection. Using a murine intratracheal infection model, our results demonstrated that CD4(+) T cells were not required for the expansion and trafficking of CD8(+) T cells to the site of infection. CD4(+) T cells were also not required for the generation of IFN-gamma-producing CD8(+) T cell effectors in the lungs. In CD4(-) mice, depletion of CD8(+) T cells resulted in increased intracellular infection of pulmonary macrophages by C. neoformans, increasing the pulmonary burden of the infection. Neutralization of IFN-gamma in CD4(-)CD8(+) mice similarly increased macrophage infection by C. neoformans, thereby blocking the protection provided by CD8(+) T cells. Altogether, these data support the hypothesis that effector CD8(+) T cell function is independent of CD4(+) T cells and that IFN-gamma production from CD8(+) T cells plays a role in controlling C. neoformans by limiting survival of C. neoformans within macrophages.

Animals↗

PubMed2005

Role of IFN-gamma in regulating T2 immunity and the development of alternatively activated macrophages during allergic bronchopulmonary mycosis.

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of a chronic pulmonary fungal infection accompanied by an "allergic" response (T2) to the infection, i.e., a model of an allergic bronchopulmonary mycosis. Our objective was to determine whether IFN-gamma plays a role in regulating the pulmonary T2 immune response in C. neoformans-infected C57BL/6 mice. Long-term pulmonary fungistasis was lost in IFN-gamma knockout (KO) mice, resulting in an increased pulmonary burden of fungi at wk 3. IFN-gamma was required for the early influx of leukocytes into the lungs but was not required later in the infection. By wk 3, eosinophil and macrophage numbers were elevated in the absence of IFN-gamma. The inducible NO synthase to arginase ratio was lower in the lungs of IFN-gamma KO mice and the macrophages had increased numbers of intracellular cryptococci and YM1 crystals, indicative of alternatively activated macrophages in these mice. There was evidence of pulmonary fibrosis in both wild-type and IFN-gamma KO mice by 5 wk postinfection. IFN-gamma production was not required for the development of T2 cytokine (IL-4, IL-5, IL-13) producing cells in the lungs and lung-associated lymph nodes or induction of an IgE response. At a number of time points, T2 cytokine production was enhanced in IFN-gamma KO mice. Thus, in the absence of IFN-gamma, C57BL/6 mice develop an augmented allergic response to C. neoformans, including enhanced generation of alternatively activated macrophages, which is accompanied by a switch from a chronic to a progressive pulmonary cryptococcal infection.

Animals↗

PubMed2005

Distinct roles for IL-4 and IL-10 in regulating T2 immunity during allergic bronchopulmonary mycosis.

Pulmonary Cryptococcus neoformans infection of C57BL/6 mice is an established model of an allergic bronchopulmonary mycosis that has also been used to test a number of immunomodulatory agents. Our objective was to determine the role of IL-4 and IL-10 in the development/manifestation of the T2 response to C. neoformans in the lungs and lung-associated lymph nodes. In contrast to wild-type (WT) mice, which develop a chronic infection, pulmonary clearance was significantly greater in IL-4 knockout (KO) and IL-10 KO mice but was not due to an up-regulation of a non-T cell effector mechanism. Pulmonary eosinophilia was absent in both IL-4 KO and IL-10 KO mice compared with WT mice. The production of IL-4, IL-5, and IL-13 by lung leukocytes from IL-4 KO and IL-10 KO mice was lower but IFN-gamma levels remained the same. TNF-alpha and IL-12 production by lung leukocytes was up-regulated in IL-10 KO but not IL-4 KO mice. Overall, IL-4 KO mice did not develop the systemic (lung-associated lymph nodes and serum) or local (lungs) T2 responses characteristic of the allergic bronchopulmonary C. neoformans infection. In contrast, the systemic T2 elements of the response remained unaltered in IL-10 KO mice whereas the T2 response in the lungs failed to develop indicating that the action of IL-10 in T cell regulation was distinct from that of IL-4. Thus, although IL-10 has been reported to down-regulate pulmonary T2 responses to isolated fungal Ags, IL-10 can augment pulmonary T2 responses if they occur in the context of fungal infection.

Animals↗

PubMed2005

CD8+ T cell contributions to allergen induced pulmonary inflammation and airway hyperreactivity.

The pathogenesis of asthma has been linked to the production of type 2 cytokines, which can be expressed by several cell types in the lung. These studies investigated CD8(+) T cell responses in a murine cockroach antigen (CRA) model of asthma. The results from these present studies show that depletion of CD8(+) T cells after allergen sensitization to CRA significantly reduces airway hyperreactivity, airway eosinophilia and pulmonary type 2 cytokine levels. The data demonstrate that CD8(+) T cells from CRA-sensitized mice can produce type 2 cytokines IL-4, IL-5 and IL-13 upon antigen challenge, and that the transfer of these cells into naive mice will cause airway hyperreactivity when exposed to CRA. We found that the transferred airway response is dependent on both IL-4 and IL-13 from CD8(+) T cells using cytokine knockout mice. Compared to CD4(+) T cells, CD8(+) T cells were not as numerous in the lungs of sensitized and challenged mice, but were as efficacious in the transfer of airway disease. The most severe airway response was observed when both CD4(+) and CD8(+) T cells were transferred at the same time. Altogether, these studies highlight a role for CD8(+) T lymphocytes in the development of allergen-induced airway responses.

Adoptive Transfer↗

PubMed2005

Development of allergic airway disease in mice following antibiotic therapy and fungal microbiota increase: role of host genetics, antigen, and interleukin-13.

Lending support to the hygiene hypothesis, epidemiological studies have demonstrated that allergic disease correlates with widespread use of antibiotics and alterations in fecal microbiota ("microflora"). Antibiotics also lead to overgrowth of the yeast Candida albicans, which can secrete potent prostaglandin-like immune response modulators, from the microbiota. We have recently developed a mouse model of antibiotic-induced gastrointestinal microbiota disruption that is characterized by stable increases in levels of gastrointestinal enteric bacteria and Candida. Using this model, we have previously demonstrated that microbiota disruption can drive the development of a CD4 T-cell-mediated airway allergic response to mold spore challenge in immunocompetent C57BL/6 mice without previous systemic antigen priming. The studies presented here address important questions concerning the universality of the model. To investigate the role of host genetics, we tested BALB/c mice. As with C57BL/6 mice, microbiota disruption promoted the development of an allergic response in the lungs of BALB/c mice upon subsequent challenge with mold spores. In addition, this allergic response required interleukin-13 (IL-13) (the response was absent in IL-13(-/-) mice). To investigate the role of antigen, we subjected mice with disrupted microbiota to intranasal challenge with ovalbumin (OVA). In the absence of systemic priming, only mice with altered microbiota developed airway allergic responses to OVA. The studies presented here demonstrate that the effects of microbiota disruption are largely independent of host genetics and the nature of the antigen and that IL-13 is required for the airway allergic response that follows microbiota disruption.

Animals↗

PubMed2005

Transient neutralization of tumor necrosis factor alpha can produce a chronic fungal infection in an immunocompetent host: potential role of immature dendritic cells.

The mechanisms underlying induction of immune dysregulation and chronic fungal infection by a transient tumor necrosis factor alpha (TNF-alpha) deficiency remain to be defined. The objective of our studies was to determine the potential contribution of neutropenia and immature dendritic cells to the immune deviation. Administration of an anti-TNF-alpha monoclonal antibody at day 0 neutralized TNF-alpha only during the first week of a pulmonary Cryptococcus neoformans infection. Transient neutralization of TNF-alpha resulted in transient depression of interleukin-12 (IL-12), monocyte chemotactic protein 1 (MCP-1), and gamma interferon (IFN-gamma) production but permanently impaired long-term clearance of the infection from the lungs even after the levels of these cytokines increased and a vigorous inflammatory response developed. Early neutrophil recruitment was defective in the absence of TNF-alpha. However, as demonstrated by neutrophil depletion studies, this did not account for the decrease in IL-12 and IFN-gamma levels and did not play a role in establishing chronic pulmonary cryptococcal infection. Transient TNF-alpha neutralization also produced a deficiency in CD11c(+) MHC II(+) cells and IL-12 in the lymph nodes, potentially implicating a defect in mature dendritic cell trafficking. Transfer of cryptococcal antigen-pulsed immature dendritic cells into naive mice prior to intratracheal challenge resulted in the development of a nonprotective immune response to C. neoformans that was similar to that observed in anti-TNF-alpha-treated mice (increased IL-4, IL-5, and IL-10 levels, pulmonary eosinophilia, and decreased clearance). Thus, stimulation of an antifungal response by immature dendritic cells can result in an immune deviation similar to that produced by transient TNF-alpha deficiency, identifying a new mechanism by which a chronic fungal infection can occur in an immunocompetent host.

Animals↗

PubMed2005

The gamma interferon receptor is required for the protective pulmonary inflammatory response to Cryptococcus neoformans.

Mice with a null deletion mutation in the gamma interferon (IFN-gamma) receptor gene were used to study the role of IFN-gamma responsiveness during experimental pulmonary cryptococcosis. Cryptococcus neoformans was inoculated intratracheally into mice lacking the IFN-gamma receptor gene (IFN-gammaR-/-) and into control mice (IFN-gammaR+/+). The numbers of CFU in lung, spleen, and brain were determined to assess clearance; cytokines produced by lung leukocytes were measured, and survival curves were generated. In the present study, we demonstrate the following points. (i) IFN-gammaR-/- mice are markedly more susceptible to C. neoformans infection than IFN-gammaR+/+ mice. (ii) In the absence of IFN-gamma signaling, pulmonary CFU continue to increase over the course of infection, and the infection disseminates to the brain. (iii) In the absence of IFN-gamma receptor, recruitment of inflammatory cells in response to pulmonary cryptococcal infection is not impaired. (iv) At week 5 postinfection, IFN-gammaR-/- mice have recruited greater numbers of leukocytes into their lungs, with neutrophils, eosinophils, and lymphocytes accounting for this cellular increase. (v) IFN-gamma signaling is required for the development of a T1 over a T2 immune response in the lung following cryptococcal infection. These results indicate that in the absence of IFN- gamma responsiveness, even though the recruitment of pulmonary inflammatory cells is not impaired and the secretion of IFN-gamma is not affected, IFN-gammaR-/- mice do not have the ability to resolve the cryptococcal infection. In conclusion, our data suggest that proper functional IFN-gamma signaling, possibly through a mechanism which inhibits the potentially disease-promoting T2 response, is required for mice to confine the cryptococcal infection.

Animals↗

PubMed2005

Immune regulation during allergic bronchopulmonary mycosis: lessons taught by two fungi.

Allergic bronchopulmonary mycosis (ABPM) is a devastating pulmonary disease that results from an aggressive allergic response to fungal colonization in the airways. Animal models using either fungal antigen or live infection reproduce most of the clinical features seen during ABPM in humans. Results from these studies have facilitated a detailed analysis of the key factors involved in the afferent as well as efferent phase of the disease. This review focuses on allergic bronchopulmonary disease caused by two different fungi (Aspergillus fumigatus and Cryptococcus neoformans): allergic bronchopulmonary aspergillosis and allergic bronchopulmonary cryptococcosis. Observations from both models underline the importance of initial innate immune responses and their translation into appropriate adaptive responses. In addition, data derived from knockout studies give emphasis to targeting cytokines and chemokines as a therapeutic strategy in the treatment of ABPM.

Animals↗

PubMed2004

Does the microbiota regulate immune responses outside the gut?

Perturbations in the gastrointestinal (GI) microbiota composition that occur as a result of antibiotics and diet in "westernized" countries are strongly associated with allergies and asthma ("hygiene hypothesis"). The microbiota ("microflora") plays a crucial role in the development of mucosal tolerance, including the airways. Significant attention has been focused on the role of the microbiota in GI development, immune adaptation and initiation of GI inflammatory diseases. This review covers the post-developmental functions that the microbiota plays in regulating immunological tolerance to allergen exposure outside the GI tract and proposes the question: is the microbiota a major regulator of the immune system?

Animals↗

PubMed2004

Novel chemokine responsiveness and mobilization of neutrophils during sepsis.

Blood neutrophils (PMN) are usually unresponsive to CC chemokines such as monacyte chemotactic protein-1 and macrophage inflammatory protein-1 alpha. In rodents, the lung buildup of PMN as determined by myeloperoxidase (MPO) activity after airway instillation of bacterial lipopolysaccharide (LPS) was independent of MCP-1 and MIP-1 alpha. In striking contrast, during sepsis following cecal ligation and puncture (CLP), blood PMN demonstrated mRNA for CC chemokine receptors. Furthermore, PMN from CLP, but not from sham rodents, bound MCP-1 and MIP-1 alpha and responded chemotactically in vitro to both MCP-1 and MIP-1 alpha. In CCR2(-/-) mice or WT mice treated in vivo with antibodies to either MCP-1 or MIP-1 alpha, MPO activity was greatly attenuated in CLP animals. In CLP mice, increased serum IL-6 levels were found to be dependent on CCR2, MCP-1, and MIP-1 alpha. When PMN from CLP rodents were incubated in vitro with either MCP-1 or MIP-1 alpha, release of IL-6 was also shown. These findings suggest that sepsis fundamentally alters the trafficking of PMN into the lung in a manner that now engages functional responses to CC chemokines.

Animals↗