Endogenous IFN-γ facilitates Pneumocystis infection and downregulates carbohydrate receptors in CD4+ T cell-depleted mice.

IFN-γ plays a critical role in host defense against intracellular pathogens. IFN-γ is produced in the bronchoalveolar lavage fluid of mice infected with Pneumocystis, but the role of IFN-γ in host defense against Pneumocystis remains controversial. It has been previously reported that although exogenous IFN-γ has beneficial effects on eradication of Pneumocystis, endogenous IFN-γ has a negative impact on innate immunity in immunocompromised hosts. Surprisingly, CD4+ T cell-depleted IFN-γ deficient (GKO) mice exhibit resistance to Pneumocystis. Alveolar macrophages (AM) from GKO mice exhibit higher expression of macrophage mannose receptor (MMR) and Dectin-1. Concomitantly, they exhibited greater ability to phagocytize Pneumocystis, and this activity was suppressed by inhibitors of these receptors. Incubation with IFN-γ resulted in a reduction in both the expression of these receptors on AM and their Pneumocystis-phagocytic activity. These results indicate that endogenous IFN-γ facilitates Pneumocystis to escape from host innate immunity by attenuating the phagocytic activity of AM via downregulation of MMR and Dectin-1.

Surfactant protein-D modulation of pulmonary macrophage phenotype is controlled by S-nitrosylation.

Surfactant protein-D (SP-D) is a regulator of pulmonary innate immunity whose oligomeric state can be altered through S-nitrosylation to regulate its signaling function in macrophages. Here, we examined how nitrosylation of SP-D alters the phenotypic response of macrophages to stimuli both in vivo and in vitro. Bronchoalveolar lavage (BAL) from C57BL6/J and SP-D-overexpressing (SP-D OE) mice was incubated with RAW264.7 cells ± LPS. LPS induces the expression of the inflammatory genes Il1b and Nos2, which is reduced 10-fold by SP-D OE-BAL. S-nitrosylation of the SP-D OE-BAL (SNO-SP-D OE-BAL) abrogated this inhibition. SNO-SP-D OE-BAL alone induced Il1b and Nos2 expression. PCR array analysis of macrophages incubated with SP-D OE-BAL (±LPS) shows increased expression of repair genes, Ccl20, Cxcl1, and Vcam1, that was accentuated by LPS. LPS increases inflammatory gene expression, Il1a, Nos2, Tnf, and Ptgs2, which was accentuated by SNO-SP-D OE-BAL but inhibited by SP-D OE-BAL. The transcription factor NF-κB was identified as a target for SNO-SP-D by IPA, which was confirmed by Trans-AM ELISA in vitro. In vivo, SP-D overexpression increases the burden of infection in a Pneumocystis model while increasing cellular recruitment. Expression of iNOS and the production of NO metabolites were significantly reduced in SP-D OE mice relative to C57BL6/J. Inflammatory gene expression was increased in infected C57BL6/J mice but decreased in SP-D OE. SP-D oligomeric structure was disrupted in C57BL6/J infected mice but unaltered within SP-D OE. Thus SP-D modulates macrophage phenotype and the balance of multimeric to trimeric SP-D is critical to this regulation.

Intrinsic Programming of Alveolar Macrophages for Protective Antifungal Innate Immunity Against Pneumocystis Infection.

Invasive fungal infections, including Pneumocystis Pneumonia (PcP), remain frequent life-threatening conditions of patients with adaptive immune defects. While innate immunity helps control pathogen growth early during infection, it is typically not sufficient for complete protection against Pneumocystis and other human fungal pathogens. Alveolar macrophages (AM) possess pattern recognition molecules capable of recognizing antigenic and structural determinants of Pneumocystis. However, this pathogen effectively evades innate immunity to infect both immunocompetent and immunosuppressed hosts, albeit with differing outcomes. During our studies of mouse models of PcP, the FVB/N strain was identified as unique because of its ability to mount a protective innate immune response against Pneumocystis infection. In contrast to other immunocompetent strains, which become transiently infected prior to the onset of adaptive immunity, FVB/N mice rapidly eradicated Pneumocystis before an adaptive immune response was triggered. Furthermore, FVB/N mice remained highly resistant to infection even in the absence of functional T cells. The effector mechanism of innate protection required the action of functional alveolar macrophages, and the adoptive transfer of resistant FVB/N AMs, but not susceptible CB.17 AMs, conferred protection to immunodeficient mice. Macrophage IFNγ receptor signaling was not required for innate resistance, and FVB/N macrophages were found to display markers of alternative activation. IFNγ reprogrammed resistant FVB/N macrophages to a permissive M1 biased phenotype through a mechanism that required direct activation of the macrophage IFNγR. These results demonstrate that appropriately programmed macrophages provide protective innate immunity against this opportunistic fungal pathogen, and suggest that modulating macrophage function may represent a feasible therapeutic strategy to enhance antifungal host defense. The identification of resistant and susceptible macrophages provides a novel platform to study not only the mechanisms of macrophage-mediated antifungal defense, but also the mechanisms by which Pneumocystis evades innate immunity.

Murine models of Pneumocystis infection recapitulate human primary immune disorders.

Despite the discovery of key pattern recognition receptors and CD4+ T cell subsets in laboratory mice, there is ongoing discussion of the value of murine models to reflect human disease. Pneumocystis is an AIDS-defining illness, in which risk of infection is inversely correlated with peripheral CD4+ T cell counts. Due to medical advances in the control of HIV, the current epidemiology of Pneumocystis infection is predominantly due to primary human immunodeficiencies and immunosuppressive therapies. To this end, we found that every human genetic immunodeficiency associated with Pneumocystis infection that has been tested in mice recapitulated susceptibility. For example, humans with a loss-of-function IL21R mutation are severely immunocompromised. We found that IL-21R, in addition to CD4+ T cell intrinsic STAT3 signaling, were required for generating protective antifungal class-switched antibody responses, as well as effector T cell-mediated protection. Furthermore, CD4+ T cell intrinsic IL-21R/STAT3 signaling was required for CD4+ T cell effector responses, including IL-22 production. Recombinant IL-22 administration to Il21r-/- mice induced the expression of a fungicidal peptide, cathelicidin antimicrobial peptide, which showed in vitro fungicidal activity. In conclusion, SPF laboratory mice faithfully replicate many aspects of human primary immunodeficiency and provide useful tools to understand the generation and nature of effector CD4+ T cell immunity.

Characterization of p57, a Stage-Specific Antigen of Pneumocystis murina.

Pneumocystis has a large multicopy gene family encoding proteins related to the major surface glycoprotein (Msg), whose functions are largely unknown. We expressed one such protein of Pneumocystis murina, p57, which is encoded by 3 highly conserved genes, and demonstrated by immunoblot that immunocompetent mice that were immunized with crude Pneumocystis antigens or that had cleared Pneumocystis infection developed antibodies to p57. Using hyperimmune anti-p57 serum combined with immunolabeling, we found that p57 was expressed by small trophic forms and intracystic bodies, whereas it was not expressed on larger trophic forms or externally by cysts. Expression of p57 and Msg by trophic forms was largely mutually exclusive. Treatment of infected animals with caspofungin inhibited cyst formation and markedly decreased p57 expression. While p57 expression was seen in immunocompetent mice infected with Pneumocystis, immunization with recombinant p57 did not result in altered cytokine expression by lymphocytes or in diminished infection in such mice. Thus, p57 appears to be a stage-specific antigen of Pneumocystis that is expressed on intracystic bodies and young trophic forms and may represent a mechanism to conserve resources in organisms during periods of limited exposure to host immune responses.

The trophic life cycle stage of Pneumocystis 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 ß-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.

Pneumocystis-Driven Inducible Bronchus-Associated Lymphoid Tissue Formation Requires Th2 and Th17 Immunity.

Inducible bronchus-associated lymphoid tissue (iBALT) is an ectopic lymphoid structure composed of highly organized T cell and B cell zones that forms in the lung in response to infectious or inflammatory stimuli. Here, we develop a model for fungal-mediated iBALT formation, using infection with Pneumocystis that induces development of pulmonary lymphoid follicles. Pneumocystis-dependent iBALT structure formation and organization required CXCL13 signaling. Cxcl13 expression was regulated by interleukin (IL)-17 family members, as Il17ra-/-, Il17rb-/-, and Il17rc-/- mice failed to develop iBALT. Interestingly, Il17rb-/- mice have intact Th17 responses, but failed to generate an anti-Pneumocystis Th2 response. Given a role for Th2 and Th17 immunity in iBALT formation, we demonstrated that primary pulmonary fibroblasts synergistically upregulated Cxcl13 transcription following dual stimulation with IL-13 and IL-17A in a STAT3/GATA3-dependent manner. Together, these findings uncover a role for Th2/Th17 cells in regulating Cxcl13 expression and provide an experimental model for fungal-driven iBALT formation.

Pneumocystis 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 TGFß 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.

Consensus guidelines for the treatment of yeast infections in the haematology, oncology and intensive care setting, 2014.

Pathogenic yeast forms are commonly associated with invasive fungal disease in the immunocompromised host, including patients with haematological malignancies and patients of haemopoietic stem cell transplants. Yeasts include the Candida spp., Cryptococcus spp., Pneumocystis jirovecii and some lesser-known pathogens. Candida species remain the most common cause of invasive yeast infections (and the most common human pathogenic fungi). These guidelines present evidence-based recommendations for the antifungal management of established, invasive yeast infections in adult and paediatric patients in the haematology/oncology setting. Consideration is also given to the critically ill patient in intensive care units, including the neonatal intensive care unit. Evidence for 'pre-emptive' or 'diagnostic-driven antifungal therapy' is also discussed. For the purposes of this paper, invasive yeast diseases are categorised under the headings of invasive candidiasis, cryptococcosis and uncommon yeast infections. Specific recommendations for the management of Pneumocystis jirovecii are presented in an accompanying article (see consensus guidelines by Cooley et al. appearing elsewhere in this supplement).

Clearance of Pneumocystis murina infection is not dependent on MyD88.

To determine if myeloid differentiation factor 88 (MyD88), which is necessary for signaling by most TLRs and IL-1Rs, is necessary for control of Pneumocystis infection, MyD88-deficient and wild-type mice were infected with Pneumocystis by exposure to infected seeder mice and were followed for up to 106 days. MyD88-deficient mice showed clearance of Pneumocystis and development of anti-Pneumocystis antibody responses with kinetics similar to wild-type mice. Based on expression levels of select genes, MyD88-deficient mice developed immune responses similar to wild-type mice. Thus, MyD88 and the upstream pathways that rely on MyD88 signaling are not required for control of Pneumocystis infection.

CD11c⁺ cells primed with unrelated antigens facilitate an accelerated immune response to influenza virus in mice.

Recent evidence suggests that an individual's unique history and sequence of exposures to pathogens and antigens may dictate downstream immune responses to disparate antigens. We show that the i.n. delivery of nonreplicative virus-like particles (VLPs), which bear structural but no antigenic similarities to respiratory pathogens, acts to prime the lungs of both C56BL/6 and BALB/c mice, facilitating heightened and accelerated primary immune responses to high-dose influenza challenge, thus providing a nonpathogenic model of innate imprinting. These responses correspond closely to those observed following natural infection with the opportunistic fungus, Pneumocystis murina, and are characterized by accelerated antigen processing by DCs and alveolar macrophages, an enhanced influx of cells to the local tracheobronchial lymph node, and early upregulation of T-cell co-stimulatory/adhesion molecules. CD11c⁺ cells, which have been directly exposed to VLPs or Pneumocystis are necessary in facilitating enhanced clearance of influenza virus, and the repopulation of the lung by Ly-6C⁺ precursors relies on CCR2 expression. Thus, immune imprinting 72 h after VLP-priming, or 2 weeks after Pneumocystis-priming is CCR2-mediated and results from the enhanced antigen processing, maturation, and trafficking abilities of DCs and alveolar macrophages, which cause accelerated influenza-specific primary immune responses and result in superior viral clearance.

MyD88 signaling regulates both host defense and immunopathogenesis during pneumocystis infection.

The immune response protects against Pneumocystis infection but is also a key component of Pneumocystis pneumonia (PcP)-related immunopathogenesis. Signaling through myeloid differentiation factor 88 (MyD88) is critical for activation of immune pathways downstream of TLRs and IL-1R. To determine whether MyD88 regulates normal host defense against Pneumocystis, nonimmunosuppressed wild-type (WT) and MyD88-deficient mice were infected. MyD88(-/-) mice had higher early Pneumocystis burdens than did WT mice but mounted an effective adaptive immune response and cleared Pneumocystis similarly to WT. However, MyD88(-/-) mice displayed a more intense and prolonged pulmonary immune response than did WT mice. To determine the role of MyD88 in the development of PcP-related immunopathogenesis, WT and MyD88(-/-) mice were rendered susceptible to PcP by depletion of CD4(+) T cells. At 4 wk postinfection, CD4-depleted WT and MyD88(-/-) mice harbored similar organism burdens, but MyD88(-/-) mice were protected from the PcP-related respiratory impairment observed in WT mice. Improved pulmonary physiology in MyD88(-/-) mice correlated with lower lung CCL2 levels and reduced cell recruitment. However, by 5 wk postinfection, the overall health of MyD88(-/-) mice began to deteriorate rapidly relative to WT, with accelerated weight loss, impaired lung function, and exacerbated alveolar inflammation. This physiological decline of MyD88(-/-) mice was associated with increased TNF-α and IFN-γ in the lung, and by the inability to control Pneumocystis burden. Thus, MyD88 is not required for resistance to Pneumocystis infection, but limits the adaptive immune response in immunocompetent mice. In the setting of active PcP, MyD88 signaling contributes to both immunopathogenesis and control of fungal burden.

Alveolar macrophages in neonatal mice are inherently unresponsive to Pneumocystis murina infection.

Pneumocystis pneumonia was first diagnosed in malnourished children and has more recently been found in children with upper respiratory symptoms. We previously reported that there is a significant delay in the immune response in newborn mice infected with Pneumocystis compared to adults (Garvy BA, Harmsen AG, Infect. Immun. 64:3987-3992, 1996, and Garvy BA, Qureshi M, J. Immunol. 165:6480-6486, 2000). This delay is characterized by the failure of neonatal lungs to upregulate proinflammatory cytokines and attract T cells into the alveoli. Here, we report that regardless of the age at which we infected the mice, they failed to mount an inflammatory response in the alveolar spaces until they were 21 days of age or older. Anti-inflammatory cytokines had some role in dampening inflammation, since interleukin-10 (IL-10)-deficient pups cleared Pneumocystis faster than wild-type pups and the neutralization of transforming growth factor beta (TGF-ß) with specific antibody enhanced T cell migration into the lungs at later time points. However, the clearance kinetics were similar to those of control pups, suggesting that there is an intrinsic deficiency in the ability of innate immunity to control Pneumocystis. We found, using an adoptive transfer strategy, that the lung environment contributes to association of Pneumocystis organisms with alveolar macrophages, implying no intrinsic deficiency in the binding of Pneumocystis by neonatal macrophages. Using both in vivo and in vitro assays, we found that Pneumocystis organisms were less able to stimulate translocation of NF-κB to the nucleus of alveolar macrophages from neonatal mice. These data indicate that there is an early unresponsiveness of neonatal alveolar macrophages to Pneumocystis infection that is both intrinsic and related to the immunosuppressive environment found in neonatal lungs.

When to start ART in the setting of acute AIDS-related opportunistic infections: the time is now!

Despite the substantial benefits of combination antiretroviral therapy (ART), a significant proportion of HIV-infected individuals still present with advanced disease and active AIDS-related opportunistic infections (OIs). The weight of evidence from recent studies supports the early initiation of ART (ie, within 2 weeks of initiating treatment for the acute OIs). Initiating ART early in acutely ill patients can reduce AIDS-related progression and death. Early ART has not been associated with increased rates of immune reconstitution inflammatory syndrome in prospective studies of non-tuberculosis OIs, although this concern is frequently cited as a reason to delay ART. Nor has early ART been associated with increased adverse outcomes. Nonetheless, initiating ART early in acute care settings can be challenging to implement and requires a well-coordinated multidisciplinary team with expertise in ART management.

Primary alveolar epithelial cell surface membrane microdomain function is required for Pneumocystis ß-glucan-induced inflammatory responses.

Intense lung inflammation characterizes respiratory failure associated with Pneumocystis pneumonia. Our laboratory has previously demonstrated that alveolar epithelial cells (AECs) elaborate inflammatory cytokines and chemokines in response to the Pneumocystis carinii cell wall constituent ß-(1→3)-glucan (PCBG), and that these responses require lactosylceramide, a prominent glycosphingolipid constituent of certain cell membrane microdomains. The relevance of membrane microdomains, also termed plasma membrane lipid rafts, in cell signaling and macromolecule handling has been increasingly recognized in many biologic systems, but their role in P. carinii-induced inflammation is unknown. To investigate the mechanisms of microdomain-dependent P. carinii-induced inflammation, we challenged primary rat AECs with PCBG with or without pre-incubation with inhibitors of microdomain function. Glycosphingolipid and cholesterol rich microdomain inhibition resulted in significant attenuation of P. carinii-induced expression of TNF-α and the rodent C-X-C chemokine MIP-2, as well as their known inflammatory secondary signaling pathways. We have previously shown that protein kinase C (PKC) is activated by PCBG challenge and herein show that PKC localizes to AEC microdomains. We also demonstrate by conventional microscopy, fluorescence microscopy, confocal microscopy and spectrophotofluorimetry that AECs internalize fluorescently-labeled PCBG by microdomain-mediated mechanisms, and that anti-microdomain pretreatments prevent internalization. Taken together, these data suggest an important role for AEC microdomain function in PCBG-induced inflammatory responses. This offers a potential novel target for therapeutics for a condition that continues to exert unacceptable morbidity and mortality among immunocompromised populations.

Pneumocystis elicits a STAT6-dependent, strain-specific innate immune response and airway hyperresponsiveness.

It is widely held that exposure to pathogens such as fungi can be an agent of comorbidity, such as exacerbation of asthma or chronic obstructive pulmonary disease. Although many studies have examined allergic responses to fungi and their effects on pulmonary function, the possible pathologic implications of the early innate responses to fungal pathogens have not been explored. We examined early responses to the atypical fungus Pneumocystis in two common strains of mice in terms of overall immunological response and related pathology, such as cell damage and airway hyperresponsiveness (AHR). We found a strong strain-specific response in BALB/c mice that included recruitment of neutrophils, NK, NKT, and CD4 T cells. This response was accompanied by elevated indicators of lung damage (bronchoalveolar lavage fluid albumin and LDH) and profound AHR. This early response was absent in C57BL/6 mice, although both strains exhibited a later response associated with the clearance of Pneumocystis. We found that this AHR could not be attributed exclusively to the presence of recruited neutrophils, NKT, NK, or CD4 cells or to the actions of IFN-γ or IL-4. However, in the absence of STAT6 signaling, AHR and inflammatory cell recruitment were virtually absent. Gene expression analysis indicated that this early response included activation of several transcription factors that could be involved in pulmonary remodeling. These results show that exposure to a fungus such as Pneumocystis can elicit pulmonary responses that may contribute to morbidity, even without prior sensitization, in the context of certain genetic backgrounds.

Pneumocystis infection and the pathogenesis of chronic obstructive pulmonary disease.

With increases in the immunocompromised patient population and aging of the HIV+ population, the risk of serious fungal infections and their complications will continue to rise. In these populations, infection with the fungal opportunistic pathogen Pneumocystis jirovecii remains a leading cause of morbidity and mortality. Infection with Pneumocystis (Pc) has been shown to be associated with the development of chronic obstructive pulmonary disease (COPD) in human subjects with and without HIV infection and in non-human primate models of HIV infection. In human studies and in a primate model of HIV/Pc co-infection, we have shown that antibody response to the Pc protein, kexin (KEX1), correlates with protection from colonization, Pc pneumonia, and COPD. These findings support the hypothesis that immunity to KEX1 may be critical to controlling Pc colonization and preventing or slowing development of COPD.

Necrotic adenoids in post-transplant lymphoproliferative disorders.

PROBLEM: Post-transplant lymphoproliferative disorders (PTLD) are a potentially fatal complication after solid organ transplantation. The majority of cases are associated with Epstein Barr virus infection (EBV). The first manifestations of PTLD are frequently observed in the ENT area with adenoidal and/or tonsillar enlargement. METHODOLOGY: We present the case of a 12-year old girl with a total nasal obstruction and tonsillitis five months after a kidney transplantation for bilateral congenital kidney hypoplasia. RESULTS: The EBV genome was detected by polymerase reaction three months after surgery. Fiberoptic examination revealed an obstructive necrotic mass in the naso-pharynx. The anatomic-pathologic analysis revealed necrotic adenoids. CONCLUSIONS: Necrotic tonsillitis is common. Necrosis of the adenoids, although rarer, can also occur and explains the important respiratory distress. Since two thirds of PTLD patients present with clinical symptoms in the ENT area, the otorhinolaryngologist should be aware of this complication.

Pneumocystis infection in an immunocompetent host can promote collateral sensitization to respiratory antigens.

Infection with the opportunistic fungal pathogen Pneumocystis is assumed to pass without persistent pathology in immunocompetent hosts. However, when immunocompetent BALB/c mice were inoculated with Pneumocystis, a vigorous Th2-like pulmonary inflammation ensued and peaked at 14 days postinfection. This coincided with a 10-fold increase in the number of antigen-presenting cells (APCs) in the lung, and these cells were capable of presenting antigen in vitro, as well as greater uptake of antigen in vivo. When mice were presented with exogenous antigen at the 14-day time point of the infection, they developed respiratory sensitization to that antigen, in the form of increased airway hyperresponsiveness upon a later challenge, whereas mice not infected but presented with antigen did not. Like other forms of collateral sensitization, this response was dependent on interleukin-4 receptor signaling. This ability to facilitate sensitization to exogenous antigen has been previously reported for other infectious disease agents; however, Pneumocystis appears to be uniquely capable in this respect, as a single intranasal dose without added adjuvant, when it was administered at the appropriate time, was sufficient to initiate sensitization. Pneumocystis infection probably occurs in most humans during the first few years of life, and in the vast majority of cases, it fails to cause any overt direct pathology. However, as we show here, Pneumocystis can be an agent of comorbidity at this time by facilitating respiratory sensitization that may relate to the later development or exacerbation of obstructive airway disease.