Overcoming Hurdles to Development of a Vaccine Against \u3cem\u3ePneumocystis jirovecii\u3c/em\u3e

Development of Pneumocystis pneumonia (PCP) is a common problem among immunosuppressed individuals. There are windows of opportunity in which vaccination would be beneficial, but to date, no vaccines have made it to clinical trials. Significant hurdles to vaccine development include host range specificity, making it difficult to translate from animal models to humans. Discovery of cross-reactive epitopes is critical to moving vaccine candidates from preclinical animal studies to clinical trials

The Life Cycle Stages of \u3cem\u3ePneumocystis murina\u3c/em\u3e Have Opposing Effects on the Immune Response to This Opportunistic Fungal Pathogen

The cell wall β-glucans of Pneumocystis cysts have been shown to stimulate immune responses in lung epithelial cells, dendritic cells, and alveolar macrophages. Little is known about how the trophic life forms, which do not have a fungal cell wall, interact with these innate immune cells. Here we report differences in the responses of both neonatal and adult mice to the trophic and cystic life cycle stages of Pneumocystis murina. The adult and neonatal immune responses to infection with Pneumocystis murina trophic forms were less robust than the responses to infection with a physiologically normal mixture of cysts and trophic forms. Cysts promoted the recruitment of nonresident innate immune cells and T and B cells into the lungs. Cysts, but not trophic forms, stimulated increased concentrations of the cytokine gamma interferon (IFN-γ) in the alveolar spaces and an increase in the percentage of CD4+ T cells that produce IFN-γ. In vitro, bone marrow-derived dendritic cells (BMDCs) stimulated with cysts produced the proinflammatory cytokines interleukin 1β (IL-1β) and IL-6. In contrast, trophic forms suppressed antigen presentation to CD4+ T cells, as well as the β-glucan-, lipoteichoic acid (LTA)-, and lipopolysaccharide (LPS)-induced production of interleukin 1β (IL-1β), IL-6, and tumor necrosis factor alpha (TNF-α) by BMDCs. The negative effects of trophic forms were not due to ligation of mannose receptor. Our results indicate that optimal innate and adaptive immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, trophic forms suppress β-glucan-induced proinflammatory responses in vitro, suggesting that the trophic forms dampen cyst-induced inflammation in vivo

Role of Phosphoinositide 3-Kinase – Akt Signaling Pathway in the Age-Related Cytokine Dysregulation in Splenic Macrophages Stimulated via TLR-2 or TLR-4 Receptors

Age-associated defects in both B-lymphocytes and macrophages in elderly result in a reduction in the efficacy of vaccines to many Gram positive bacteria like Streptococcus pneumoniae. Splenic macrophages from aged mice have been shown to have a defect in production of pro-inflammatory cytokines (IL-6, IL-12, IL-1β, TNF-α) but exhibit increased production of IL-10 upon TLR4 ligation. Here we showed that aged macrophages demonstrate similar cytokine dysregulation phenotype upon stimulation with TLR2 ligands, or killed S. pneumoniae. We hypothesized that an age-associated increase in activity of phosphatidyl inositol 3-kinase (PI3K)-Akt signaling pathway may be playing a causal role in the age-associated cytokine dysregulation. We found that gene expression of both the regulatory (p85β) and the catalytic (p110δ) subunits of Class IA PI3K is higher in aged than in young splenic macrophages. The age-associated increase in the activity of PI3K was also demonstrated by an upregulation of P-Akt and its downstream target, glycogen synthase kinase-3 (GSK-3). Inhibition of PI3K enhanced induction of pro-inflammatory cytokines, by TLR-2/TLR-1, TLR-2/TLR-6 and TLR-4 ligands as well as heat killed S. pneumoniae (HKSP). Therefore, targeting PI3-Kinase could rescue cytokine dysregulation in aged macrophages and enhance the relevant pro-inflammatory cytokines needed to support B-cell activation and differentiation

Tuberculosis and Cardiovascular Disease: Linking the Epidemics

The burden of tuberculosis and cardiovascular disease (CVD) is enormous worldwide. CVD rates are rapidly increasing in low- and middle-income countries. Public health programs have been challenged with the overlapping tuberculosis and CVD epidemics. Monocyte/macrophages, lymphocytes and cytokines involved in cellular mediated immune responses against Mycobacterium tuberculosis are also main drivers of atherogenesis, suggesting a potential pathogenic role of tuberculosis in CVD via mechanisms that have been described for other pathogens that establish chronic infection and latency. Studies have shown a pro-atherogenic effect of antibody-mediated responses against mycobacterial heat shock protein-65 through cross reaction with self-antigens in human vessels. Furthermore, subsets of mycobacteria actively replicate during latent tuberculosis infection (LTBI), and recent studies suggest that LTBI is associated with persistent chronic inflammation that may lead to CVD. Recent epidemiologic work has shown that the risk of CVD in persons who develop tuberculosis is higher than in persons without a history of tuberculosis, even several years after recovery from tuberculosis. Together, these data suggest that tuberculosis may play a role in the pathogenesis of CVD. Further research to investigate a potential link between tuberculosis and CVD is warranted

Bias of the Immune Response to \u3ci\u3ePneumocystis murina\u3c/i\u3e Does Not Alter the Ability of Neonatal Mice to Clear the Infection

Newborn mice are unable to clear Pneumocystis (PC) infection with the same efficiency as adults due, in part, to their inability to develop a robust immune response to infection until three weeks of age. It is known that infants tend develop a Th2 skewed response to antigen so we sought to determine whether a biased cytokine response altered the clearance of PC infection in neonatal mice. P. murina infection in neonatal mice resulted in increased IL-4 expression by CD4 T cells and myeloid cells, augmented IL-13 secretion within the airways and increased arginase activity in the airways, indicative of Th2-type responses. P. murina-infected IL-4Rα−/− neonates had a shift towards Th1 cytokine production and increased numbers of CD4 and CD8 T cells within the lung as well as elevated levels of P. murina-specific IgG. IFNγ−/− and IL-23 p19−/− mice had altered CD4-T cell-dependent cytokine and cell responses. Though we could alter the T helper cell environment in neonatal knockout mice, there was no loss in the ability of these pups to clear infection. It is possible that the Th2 phenotype normally seen in neonatal mice protects the developing lung from pro-inflammatory immune responses without compromising host defense against P. murina

The Trophic Life Cycle Stage of the Opportunistic Fungal Pathogen \u3cem\u3ePneumocystis murina\u3c/em\u3e Hinders the Ability of Dendritic Cells to Stimulate CD4\u3csup\u3e+\u3c/sup\u3e T Cell Responses

The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including β-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with β-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells

Tuberculosis and Risk of Acute Myocardial Infarction: A Propensity Score-Matched Analysis

Several pathogens have been associated with increased cardiovascular disease (CVD) risk. Whether this occurs with Mycobacterium tuberculosis infection is unclear. We assessed if tuberculosis disease increased the risk of acute myocardial infarction (AMI). We identified patients with tuberculosis index claims from a large de-identified database of ~15 million adults enrolled in a U.S. commercial insurance policy between 2008 and 2010. Tuberculosis patients were 1:1 matched to patients without tuberculosis claims using propensity scores. We compared the occurrence of index AMI claims between the tuberculosis and non-tuberculosis cohorts using Kaplan–Meier curves and Cox Proportional Hazard models. Data on 2026 patients with tuberculosis and 2026 propensity-matched patients without tuberculosis were included. AMI was more frequent in the tuberculosis cohort compared with the non-tuberculosis cohort, 67 (3·3%) vs. 32 (1·6%) AMI cases, respectively, P \u3c 0·01. Tuberculosis was associated with an increased risk of AMI (adjusted hazard ratio (HR) 1·98, 95% confidence intervals (CI) 1·3–3·0). The results were similar when the analysis was restricted to pulmonary tuberculosis (adjusted HR 2·43, 95% CI 1·5–4·1). Tuberculosis was associated with an increased risk of AMI. CVD risk assessment should be considered in tuberculosis patients. Mechanistic studies of tuberculosis and CVD are warranted

B Lymphocytes Are Required during the Early Priming of CD4\u3csup\u3e+\u3c/sup\u3e T Cells for Clearance of \u3cem\u3ePneumocystis\u3c/em\u3e Infection in Mice

B cells play a critical role in the clearance of Pneumocystis. In addition to production of Pneumocystis-specific Abs, B cells are required during the priming phase for CD4+ T cells to expand normally and generate memory. Clearance of Pneumocystis was found to be dependent on Ag specific B cells and on the ability of B cells to secrete Pneumocystis-specific Ab, as mice with B cells defective in these functions or with a restricted BCR were unable to control Pneumocystis infection. Because Pneumocystis-specific antiserum was only able to partially protect B cell–deficient mice from infection, we hypothesized that optimal T cell priming requires fully functional B cells. Using adoptive transfer and B cell depletion strategies, we determined that optimal priming of CD4+ T cells requires B cells during the first 2–3 d of infection and that this was independent of the production of Ab. T cells that were removed from Pneumocystis-infected mice during the priming phase were fully functional and able to clear Pneumocystis infection upon adoptive transfer into Rag1−/− hosts, but this effect was ablated in mice that lacked fully functional B cells. Our results indicate that T cell priming requires a complete environment of Ag presentation and activation signals to become fully functional in this model of Pneumocystis infection

\u3cem\u3ePneumocystis\u3c/em\u3e Infection Alters the Activation State of Pulmonary Macrophages

Recent studies show a substantial incidence of Pneumocystis jirovecii colonization and infection in patients with chronic inflammatory lung conditions. However, little is known about the impact of Pneumocystis upon the regulation of pulmonary immunity. We demonstrate here that Pneumocystis polarizes macrophages towards an alternatively activated macrophage-like phenotype. Genetically engineered mice that lack the ability to signal through IL-4 and IL-13 were used to show that Pneumocystis alternative macrophage activation is dependent upon signaling through these cytokines. To determine whether Pneumocystis-induced macrophage polarization would impact subsequent immune responses, we infected mice with Pneumocystis and then challenged them with Pseudomonas aeruginosa 14 days later. In co-infected animals, a higher proportion of macrophages in the alveolar and interstitial spaces expressed both classical and alternatively activated markers and produced the regulatory cytokines TGFβ and IL-10, as well as higher arginase levels than in mice infected with P. aeruginosa alone. Our results suggest that Pneumocystis reprograms the overall macrophage repertoire in the lung to that of a more alternatively-activated setpoint, thereby altering subsequent immune responses. These data may help to explain the association between Pneumocystis infection and decline in pulmonary function

The Trophic Life Cycle Stage of \u3cem\u3ePneumocystis\u3c/em\u3e Species Induces Protective Adaptive Responses without Inflammation-Mediated Progression to Pneumonia

Pneumocystis species are fungal pathogens that cause pneumonia in immunocompromised hosts. Lung damage during Pneumocystis pneumonia is predominately due to the inflammatory immune response. Pneumocystis species have a biphasic life cycle. Optimal innate immune responses to Pneumocystis species are dependent on stimulation with the cyst life cycle stage. Conversely, the trophic life cycle stage broadly suppresses proinflammatory responses to multiple pathogen-associated molecular patterns (PAMPs), including β-1,3-glucan. Little is known about the contribution of these life cycle stages to the development of protective adaptive responses to Pneumocystis infection. Here we report that CD4+ T cells primed in the presence of trophic forms are sufficient to mediate clearance of trophic forms and cysts. In addition, primary infection with trophic forms is sufficient to prime B-cell memory responses capable of clearing a secondary infection with Pneumocystis following CD4+ T cell depletion. While trophic forms are sufficient for initiation of adaptive immune responses in immunocompetent mice, infection of immunocompromised recombination-activating gene 2 knockout (RAG2−/−) mice with trophic forms in the absence of cysts does not lead to the severe weight loss and infiltration of innate immune cells associated with the development of Pneumocystis pneumonia