Evidence for Proinflammatory β-1,6 Glucans in the Pneumocystis carinii Cell Wall

Inflammation is a major cause of respiratory impairment during Pneumocystis pneumonia. Studies support a significant role for cell wall β-glucans in stimulating inflammatory responses. Fungal β-glucans are comprised of d-glucose homopolymers containing β-1,3-linked glucose backbones with β-1,6-linked glucose side chains. Prior studies in Pneumocystis carinii have characterized β-1,3 glucan components of the organism. However, recent investigations in other organisms support important roles for β-1,6 glucans, predominantly in mediating host cellular activation. Accordingly, we sought to characterize β-1,6 glucans in the cell wall of Pneumocystis and to establish their activity in lung cell inflammation. Immune staining revealed specific β-1,6 localization in P. carinii cyst walls. Homology-based cloning facilitated characterization of a functional P. carinii kre6 (Pckre6) β-1,6 glucan synthase in Pneumocystis that, when expressed in kre6-deficient Saccharomyces cerevisiae, restored cell wall stability. Recently synthesized β-1,6 glucan synthase inhibitors decreased the ability of isolated P. carinii preparations to generate β-1,6 carbohydrate. In addition, isolated β-1,6 glucan fractions from Pneumocystis elicited vigorous tumor necrosis factor alpha (TNF-α) responses from macrophages. These inflammatory responses were significantly dampened by inhibition of host cell plasma membrane microdomain function. Together, these studies indicate that β-1,6 glucans are present in the P. carinii cell wall and contribute to lung cell inflammatory activation during infection.     

Masking of β(1-3)-Glucan in the Cell Wall of Candida albicans from Detection by Innate Immune Cells Depends on Phosphatidylserine

The virulence of Candida albicans in a mouse model of invasive candidiasis is dependent on the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). Disruption of the PS synthase gene CHO1 (i.e., cho1Δ/Δ) eliminates PS and blocks the de novo pathway for PE biosynthesis. In addition, the cho1Δ/Δ mutant''s ability to cause invasive disease is severely compromised. The cho1Δ/Δ mutant also exhibits cell wall defects, and in this study, it was determined that loss of PS results in decreased masking of cell wall β(1-3)-glucan from the immune system. In wild-type C. albicans, the outer mannan layer of the wall masks the inner layer of β(1-3)-glucan from exposure and detection by innate immune effector molecules like the C-type signaling lectin Dectin-1, which is found on macrophages, neutrophils, and dendritic cells. The cho1Δ/Δ mutant exhibits increases in exposure of β(1-3)-glucan, which leads to greater binding by Dectin-1 in both yeast and hyphal forms. The unmasking of β(1-3)-glucan also results in increased elicitation of TNF-α from macrophages in a Dectin-1-dependent manner. The role of phospholipids in fungal pathogenesis is an emerging field, and this is the first study showing that loss of PS in C. albicans results in decreased masking of β(1-3)-glucan, which may contribute to our understanding of fungus-host interactions.     

Abolishing Cell Wall Glycosylphosphatidylinositol-Anchored Proteins in Candida albicans Enhances Recognition by Host Dectin-1

Fungi can shield surface pathogen-associated molecular patterns (PAMPs) for evading host immune attack. The most common and opportunistic human pathogen, Candida albicans, can shield β-(1 3)-glucan on the cell wall, one of the major PAMPs, to avoid host phagocyte Dectin-1 recognition. The way to interfere in the shielding process for more effective antifungal defense is not well established. In this study, we found that deletion of the C. albicans GPI7 gene, which was responsible for adding ethanolaminephosphate to the second mannose in glycosylphosphatidylinositol (GPI) biosynthesis, could block the attachment of most GPI-anchored cell wall proteins (GPI-CWPs) to the cell wall and subsequently unmask the concealed β-(1,3)-glucan. Neutrophils could kill the uncloaked gpi7 mutant more efficiently with an augmented respiratory burst. The gpi7 mutant also stimulated Dectin-1-dependent immune responses of macrophages, including activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways and secretion of specific cytokines, such as tumor necrosis factor alpha (TNF-α), interleukin 6 (IL-6), and IL-12p40. Furthermore, the gpi7 null mutant could induce an enhanced inflammatory response through promoting significant recruitment of neutrophils and monocytes and could stimulate stronger Th1 and Th17 cell responses to fungal infections in vivo. These in vivo phenotypes also were Dectin-1 dependent. Thus, we assume that GPI-CWPs are involved in the immune mechanism of C. albicans escaping from host recognition by Dectin-1. Our studies also indicate that the blockage of GPI anchor synthesis is a strategy to inhibit C. albicans evading host recognition.     

Characterization of a Distinct Host Response Profile to Pneumocystis murina Asci during Clearance of Pneumocystis Pneumonia

Pneumocystis spp. are yeast-like fungi that cause pneumocystis pneumonia (PcP) in immunocompromised individuals and exacerbate chronic lung diseases in immunocompetent individuals. The Pneumocystis life cycle includes trophic forms and asci (cyst forms). The cell walls of Pneumocystis asci contain β-1,3-d-glucan, and treatment of PcP with β-1,3-d-glucan synthase inhibitors, such as anidulafungin, results in depletion of asci, but not trophic forms. The pulmonary host response during immune reconstitution (IR)-mediated clearance of PcP in anidulafungin-treated and untreated mice was characterized to identify ascus-specific responses. During IR, similar numbers of trophic forms were present in the anidulafungin-treated and untreated mice; however, asci were only present in the untreated mice. IR resulted in a significant reduction of trophic forms from the lungs in both groups and asci in the untreated group. The presence of asci in untreated mice correlated with increased β-glucan content in the lungs. The untreated mice mounted immune responses associated with a deleterious host inflammatory response, including increased CD8⁺ T cell influx and expression of macrophage inflammatory response markers. A more robust cellular response was also observed in the untreated mice, with increased numbers of macrophages and neutrophils that were associated with greater lung damage. Markers of a Th17 response were also elevated in the untreated mice. These results suggest that the host mounts unique responses to asci and trophic forms. That these 2 life cycle stages provoked distinct host response profiles has significant implications for clearance and interpretation of the host immune responses to PcP.     

The Absence of Hck,Fgr, and Lyn Tyrosine Kinases Augments Lung Innate Immune Responses to Pneumocystis murina

Src family tyrosine kinases (SFKs) phosphorylate immunotyrosine activation motifs in the cytoplasmic tail of multiple immunoreceptors, leading to the initiation of cellular effector functions, such as phagocytosis, reactive oxygen species production, and cytokine production. SFKs also play important roles in regulating these responses through the activation of immunotyrosine inhibitory motif-containing inhibitory receptors. As myeloid cells preferentially express the SFKs Hck, Fgr, and Lyn, we questioned the role of these kinases in innate immune responses to Pneumocystis murina. Increased phosphorylation of Hck was readily detectable in alveolar macrophages after stimulation with P. murina. We further observed decreased phosphorylation of Lyn on its C-terminal inhibitory tyrosine in P. murina-stimulated alveolar macrophages, indicating that SFKs were activated in alveolar macrophages in response to P. murina. Mice deficient in Hck, Fgr, and Lyn exhibited augmented clearance 3 and 7 days after intratracheal administration of P. murina, which correlated with elevated levels of interleukin 1β (IL-1β), IL-6, CXCL1/KC, CCL2/monocyte chemoattractant protein 1, and granulocyte colony-stimulating factor in lung homogenates and a dramatic increase in macrophage and neutrophil recruitment. Augmented P. murina clearance was also observed in Lyn^−/− mice 3 days postchallenge, although the level was less than that observed in Hck^−/− Fgr^−/− Lyn^−/− mice. A correlate to augmented clearance of P. murina in Hck^−/− Fgr^−/− Lyn^−/− mice was a greater ability of alveolar macrophages from these mice to kill P. murina in vitro, suggesting that SFKs regulate the alveolar macrophage effector function against P. murina. Mice deficient in paired immunoglobulin receptor B (PIR-B), an inhibitory receptor activated by SFKs, did not exhibit enhanced inflammatory responsiveness to or clearance of P. murina. Our results suggest that SFKs regulate innate lung responses to P. murina in a PIR-B-independent manner.Although the advent of prophylactic therapy against Pneumocystis jirovecii alone or in combination with highly active antiretroviral therapy has reduced the incidence of pneumonia caused by this fungal pathogen, this infection remains the most prevalent opportunistic infection in individuals with AIDS (25). Furthermore, individuals receiving immunosuppressive therapies, such as individuals undergoing solid organ or hematopoietic cell transplantation, are a growing population susceptible to Pneumocystis pneumonia (34) (33). These observations warrant a more thorough examination of interactions between Pneumocystis and the host in order to define and develop new vaccine and immunotherapeutic approaches to treat Pneumocystis pneumonia.An intense area of research over the last decade is investigating how lung immune cells recognize and respond to inhaled pathogens, such as P. murina. After inhalation of P. murina into the lungs, one of the first interactions with the host is recognition by the alveolar macrophage. Our laboratory has previously reported that alveolar macrophages recognize P. murina via the beta-glucan receptor dectin-1 (37). Recognition by dectin-1 leads to internalization of P. murina and subsequent killing of the organism, as well as elaboration of the neutrophil-attracting chemokine CXCL2/MIP-2 (37). Toll-like receptor 2 (TLR2) is an additional receptor expressed by alveolar macrophages that mediates CXCL2/MIP-2 production (54) and, in humans, interleukin-8 (IL-8) production in cooperation with the macrophage mannose receptor (39). P. murina infection in TLR2^−/− mice is prolonged and associated with a lack of inflammatory responsiveness (51). Other studies have implicated TLR4 in alveolar macrophage recognition of P. murina (6).Immunoreceptors expressed by cells of the innate immune system are abundant and fall into many different categories, such as scavenger receptors, integrins, immunoglobulin (Ig) superfamily receptors, C-type lectin receptors, and TLRs (41). As expected based on this diversity, immunoreceptor signaling in innate cells is a complex process that differs greatly for different types of receptors. For example, receptors in the C-type lectin family often utilize an immunotyrosine activation motif (ITAM) in the cytoplasmic tail for signaling (2). Receptors in the Ig superfamily, such as SIRPα and Siglec3, contain immunotyrosine inhibitory motifs (ITIMs) that initiate regulatory signals, whereas members of the TLR family signal through multiple intermediate proteins, such as TIRAP, TRIF, and MyD88 (28). One commonality in innate immunoreceptor signaling is the role of Src family tyrosine kinases (SFKs). Our current understanding of how SFKs function is due in large part to studies that have characterized ITAM-associated Fc receptor (FcγR) signaling (32). In FcγR signaling, SFKs phosphorylate two tyrosine residues in the ITAM domain, which leads to the recruitment of Syk and subsequent activation of cellular responses, such as phagocytosis and cytokine and chemokine production (32).An equally important function of SFKs is to phosphorylate ITIMs, which leads to the recruitment of SHP-1 or SHIP-1 phosphatases and subsequent regulation or inhibition of responses (21). The phosphorylation of ITIMs by SFKs is often responsible for the regulation of responses initiated by many types of immunoreceptors. Mice deficient in the SFKs Hck and Fgr have enhanced chemokine receptor signaling as a result of lower phosphorylation of the ITIM in the inhibitory receptor paired Ig receptor B (PIR-B) (55). Mice deficient in PIR-B were also found to be hyperresponsive to chemokine signaling (55). PIR-B ITIM activation has also been shown to regulate TLR-mediated macrophage responses to some gram-positive and gram-negative bacteria (26). Other studies have shown that the SFK Lyn phosphorylates an ITIM of platelet endothelial cell adhesion molecule 1 in mast cells, leading to regulated FcɛRI responses (45).Macrophage-mediated recognition of P. murina involves a variety of receptors that may be dependent on SFKs for induction as well as the regulation of responses; therefore, we sought to investigate the role of SFKs in innate immune responses to P. murina. In this study, we made the surprising observation that deficiency of Hck, Fgr, and Lyn resulted in paradoxically augmented lung clearance of P. murina and enhanced cytokine and chemokine production. This response was not due to impaired PIR-B inhibitory responses as PIR-B^−/− mice were not capable of enhanced P. murina lung clearance. We propose that the SFKs Hck, Fgr, and Lyn regulate innate immune responses to P. murina and that, therefore, novel therapeutics to control the activity of SFKs may be beneficial in treating pulmonary infections.     

Neither Classical nor Alternative Macrophage Activation Is Required for Pneumocystis Clearance during Immune Reconstitution Inflammatory Syndrome

Pneumocystis is a respiratory fungal pathogen that causes pneumonia (Pneumocystis pneumonia [PcP]) in immunocompromised patients. Alveolar macrophages are critical effectors for CD4⁺ T cell-dependent clearance of Pneumocystis, and previous studies found that alternative macrophage activation accelerates fungal clearance during PcP-related immune reconstitution inflammatory syndrome (IRIS). However, the requirement for either classically or alternatively activated macrophages for Pneumocystis clearance has not been determined. Therefore, RAG2^−/− mice lacking either the interferon gamma (IFN-γ) receptor (IFN-γR) or interleukin 4 receptor alpha (IL-4Rα) were infected with Pneumocystis. These mice were then immune reconstituted with wild-type lymphocytes to preserve the normal T helper response while preventing downstream effects of Th1 or Th2 effector cytokines on macrophage polarization. As expected, RAG2^−/− mice developed severe disease but effectively cleared Pneumocystis and resolved IRIS. Neither RAG/IFN-γR^−/− nor RAG/IL-4Rα^−/− mice displayed impaired Pneumocystis clearance. However, RAG/IFN-γR^−/− mice developed a dysregulated immune response, with exacerbated IRIS and greater pulmonary function deficits than those in RAG2 and RAG/IL-4Rα^−/− mice. RAG/IFN-γR^−/− mice had elevated numbers of lung CD4⁺ T cells, neutrophils, eosinophils, and NK cells but severely depressed numbers of lung CD8⁺ T suppressor cells. Impaired lung CD8⁺ T cell responses in RAG/IFN-γR^−/− mice were associated with elevated lung IFN-γ levels, and neutralization of IFN-γ restored the CD8 response. These data demonstrate that restricting the ability of macrophages to polarize in response to Th1 or Th2 cytokines does not impair Pneumocystis clearance. However, a cell type-specific IFN-γ/IFN-γR-dependent mechanism regulates CD8⁺ T suppressor cell recruitment, limits immunopathogenesis, preserves lung function, and enhances the resolution of PcP-related IRIS.     

Pneumocystis murina infection and cigarette smoke exposure interact to cause increased organism burden, development of airspace enlargement, and pulmonary inflammation in mice

Chronic obstructive pulmonary disease (COPD) is characterized by the presence of airflow obstruction and lung destruction with airspace enlargement. In addition to cigarette smoking, respiratory pathogens play a role in pathogenesis, but specific organisms are not always identified. Recent reports demonstrate associations between the detection of Pneumocystis jirovecii DNA in lung specimens or respiratory secretions and the presence of emphysema in COPD patients. Additionally, human immunodeficiency virus-infected individuals who smoke cigarettes develop early emphysema, but a role for P. jirovecii in pathogenesis remains speculative. We developed a new experimental model using immunocompetent mice to test the interaction of cigarette smoke exposure and environmentally acquired Pneumocystis murina infection in vivo. We hypothesized that cigarette smoke and P. murina would interact to cause increases in total lung capacity, airspace enlargement, and pulmonary inflammation. We found that exposure to cigarette smoke significantly increases the lung organism burden of P. murina. Pulmonary infection with P. murina, combined with cigarette smoke exposure, results in changes in pulmonary function and airspace enlargement characteristic of pulmonary emphysema. P. murina and cigarette smoke exposure interact to cause increased lung inflammatory cell accumulation. These findings establish a novel animal model system to explore the role of Pneumocystis species in the pathogenesis of COPD.     

Treatment with Interleukin-7 Restores Host Defense against Pneumocystis in CD4+ T-Lymphocyte-Depleted Mice

Pneumocystis pneumonia (PCP) is a major cause of morbidity and mortality in patients with HIV infection. CD4⁺ T lymphocytes are critical for host defense against this infection, but in the absence of CD4⁺ T lymphocytes, CD8⁺ T lymphocytes may provide limited host defense. The cytokine interleukin-7 (IL-7) functions to enhance lymphocyte proliferation, survival, and recruitment of immune cells to sites of infection. However, there is little known about the role of IL-7 in PCP or its potential use as an immunotherapeutic agent. We hypothesized that treatment with recombinant human IL-7 (rhIL-7) would augment host defense against Pneumocystis and accelerate pathogen clearance in CD4-depleted mice. Control and CD4-depleted mice were infected with Pneumocystis, and rhIL-7 was administered via intraperitoneal injection. Our studies indicate that endogenous murine IL-7 is part of the normal host response to Pneumocystis murina and that administration of rhIL-7 markedly enhanced clearance of Pneumocystis in CD4-depleted mice. Additionally, we observed increased recruitment of CD8⁺ T lymphocytes to the lungs and decreased apoptosis of pulmonary CD8⁺ T lymphocytes in rhIL-7-treated animals compared to those in untreated mice. The antiapoptotic effect of rhIL-7 was associated with increased levels of Bcl-2 protein in T lymphocytes. rhIL-7 immunotherapy in CD4-depleted mice also increased the number of gamma interferon (IFN-γ)-positive CD8⁺ central memory T lymphocytes in the lungs. We conclude that rhIL-7 has a potent therapeutic effect in the treatment of murine Pneumocystis pneumonia in CD4-depleted mice. This therapeutic effect is mediated through enhanced recruitment of CD8⁺ T cells and decreased apoptosis of lung T lymphocytes, with a preferential action on central memory CD8⁺ T lymphocytes.     

Evasion of innate immune responses: evidence for mannose binding lectin inhibition of tumor necrosis factor alpha production by macrophages in response to Blastomyces dermatitidis

Serum factors, including mannose binding lectins (MBL), influence innate responses to microbes. Little is known about the effects of serum factors or MBL on the interaction of Blastomyces dermatitidis, a pulmonary fungal pathogen, with macrophages or on tumor necrosis factor alpha (TNF-α) production. Since macrophage production of TNF-α is an important innate immune response, we examined a mouse peritoneal macrophage (PM) cell line (RAW) and resident PM from CD-1 mice to study TNF-α production by PM stimulated with heat-killed (HK) or live B. dermatitidis yeast cells. Mouse serum and heat-inactivated mouse serum inhibited TNF-α production 94% when macrophages were stimulated by B. dermatitidis, whereas mouse immunoglobulin G (IgG) did not have this effect. HK B. dermatitidis incubated with serum and then washed also failed to stimulate significant TNF-α production by PM. By the sandwich immunofluorescent antibody (IFA) method with anti-mouse MBL (MBL-A or -C), we showed that serum MBL bound to B. dermatitidis. When serum was absorbed with HK B. dermatitidis or live B. dermatitidis, absorbed serum failed to significantly inhibit TNF-α production by RAW cells plus B. dermatitidis, and immunoblotting showed that absorbed serum was depleted of MBL-C. If serum was absorbed with live B. dermatitidis, unbound serum was eluted, and bound serum factor(s) (BS) was released with guanidine buffer, BS inhibited TNF-α production by PM plus B. dermatitidis in a concentration-dependent manner. BS contained MBL-C, which bound B. dermatitidis, as shown by IFA assay. 1,3-β-Glucan stimulated TNF-α production by PM, and this was inhibited by mouse serum. Treatment of B. dermatitidis with anti-1,3-β-glucan antibody inhibited TNF-α production by PM. With anti-1,3-β-glucan antibody, we showed by IFA assay that B. dermatitidis contained 1,3-β-glucan. In an IFA study with B. dermatitidis, serum with an anti-mouse IgG conjugate did not result in fluorescence, yet serum blocked IFA staining of B. dermatitidis by anti-1,3-β-glucan IgG antibody. This indicated that non-IgG serum factors binding to B. dermatitidis prevented access to 1,3-β-glucan by anti-1,3-β-glucan antibody. These results suggest that the mechanism of inhibition of the innate proinflammatory immune response of PM to B. dermatitidis is mediated by serum MBL binding to B. dermatitidis at 1,3-β-glucan sites or sterically masking 1,3-β-glucan sites, thus preventing 1,3-β-glucan stimulation of PM for TNF-α production.     

Distinct Patterns of Dendritic Cell Cytokine Release Stimulated by Fungal β-Glucans and Toll-Like Receptor Agonists

β-Glucans derived from fungal cell walls have potential uses as immunomodulating agents and vaccine adjuvants. Yeast glucan particles (YGPs) are highly purified Saccharomyces cerevisiae cell walls composed of β1,6-branched β1,3-d-glucan and free of mannans. YGPs stimulated secretion of the proinflammatory cytokine tumor necrosis factor alpha (TNF-α) in wild-type murine bone marrow-derived myeloid dendritic cells (BMDCs) but did not stimulate interleukin-12p70 (IL-12p70) production. A purified soluble β1,6-branched β1,3-d-glucan, scleroglucan, also stimulated TNF-α in BMDCs. These two β-glucans failed to stimulate TNF-α in Dectin-1 (β-glucan receptor) knockout BMDCs. Costimulation of wild-type BMDCs with β-glucans and specific Toll-like receptor (TLR) ligands resulted in greatly enhanced TNF-α production but decreased IL-12p70 production compared with TLR agonists alone. The upregulation of TNF-α and downregulation of IL-12p70 required Dectin-1, but not IL-10. Gamma interferon (IFN-γ) priming did not overcome IL-12p70 reduction by β-glucans. Similar patterns of cytokine regulation were observed in human monocyte-derived dendritic cells (DCs) costimulated with YGPs and the TLR4 ligand lipopolysaccharide. Finally, costimulation of BMDCs with YGPs and either the TLR9 ligand, CpG, or the TLR2/1 ligand, Pam₃CSK₄, resulted in upregulated secretion of IL-1α and IL-10 and downregulated secretion of IL-1β, IL-6, and IFN-γ-inducible protein 10 but had no significant effects on IL-12p40, keratinocyte-derived chemokine, monocyte chemotactic protein 1, or macrophage inflammatory protein α, compared with the TLR ligand alone. Thus, β-glucans have distinct effects on cytokine responses following DC stimulation with different TLR agonists. These patterns of response might contribute to the skewing of immune responses during mycotic infections and have implications for the design of immunomodulators and vaccines containing β-glucans.The immune response following an encounter with a microbe is heavily influenced by interactions between host pattern recognition receptors (PRRs) and pathogen-associated molecular patterns (PAMPs) (28). β-Glucans, especially β-(1-3)-glucans, are the major component of the cell walls of most fungi (5). Indeed, β-glucans comprise up to 60% of the dry weight of the fungal cell wall and are considered to be a major PAMP involved in host-fungus interactions (48). Dectin-1, the major receptor for β-glucans, is a C-type lectin that is highly expressed on dendritic cells (DCs). Its expression can also be detected in macrophages, monocytes, and neutrophils (6, 45). The cytoplasmic tail of Dectin-1 contains an immunoreceptor tyrosine-based activation (ITAM)-like motif (1). Upon binding of β-glucan to Dectin-1''s extracellular lectin binding domain, the tyrosine residue within the cytoplasmic ITAM motif is phosphorylated (21, 38). This results in recruitment of spleen tyrosine kinase (Syk) and caspase recruitment domain protein 9 (Card9) (19). Although the exact signal transduction pathways have not been elucidated, these events can lead to activation of nuclear factor of activated T cells (NFAT) (16), mitogen-activated protein kinases (11), and nuclear factor kappa B (NF-κB) (2, 9, 18, 38, 42, 53), leading to cytokine production.In addition to Dectin-1, other receptors have been reported to bind β-glucans, including complement receptor 3 (CR3, a heterodimer of CD11b and CD18), lactosylceramide, scavenger receptors, and CD5 (12, 49). Host PRRs also recognize other fungal components: mannose receptor (MR) and dendritic cell intercellular adhesion molecule 3 (ICAM3)-grabbing nonintegrin (DC-SIGN) recognize mannans (30), Toll-like receptor 2 (TLR2) recognizes phospholipomannan (25), TLR4 recognizes O-linked mannans (35), and TLR9 detects fungal DNA (33, 37). During the course of a fungal infection, multiple PAMPs are likely to be stimulated by host PRRs. The final response will depend not only on the relative degree of stimulation of the individual receptors but also on whether receptor costimulation is additive, synergistic, or antagonistic. Such considerations may also be critical for vaccine design.Here, we examined the ability of purified fungal β-glucans, alone and in combination with TLR agonists, to stimulate cytokine production in mouse and human DCs. To mimic the range of β-glucans encountered during a fungal infection, both particulate and soluble β-glucans were studied. Yeast glucan particles (YGPs) derived from Saccharomyces cerevisiae are composed of β1,6-branched β1,3-d-glucan and served as the particulate β-glucan. Scleroglucan (SCG), a linear β-1,3-glucan with one β-1,6-d-glucose side chain every three residues, served as the soluble stimulus. We found that the combination of β-glucans and TLR agonists had inhibitory, neutral, or stimulatory effects depending upon the individual cytokine studied. The information gathered here has important implications for our understanding of how immune responses to fungal pathogens develop, as well as for the design of vaccines and immunomodulators containing β-glucans.     

B Cell Production of Tumor Necrosis Factor in Response to Pneumocystis murina Infection in Mice

Pneumocystis species are opportunistic fungal pathogens that induce tumor necrosis factor (TNF) production by alveolar macrophages. Here we report that B cells from the draining lymph nodes as well as lung CD4⁺ T cells are important producers of TNF upon Pneumocystis murina infection. To determine the importance of B cell-derived TNF in the primary response to P. murina, we generated bone marrow chimeras whose B cells were unable to produce TNF. The lung P. murina burden at 10 days postinfection in TNF knockout (TNFKO) chimeras was significantly higher than that in wild-type (WT) chimeras, which corresponded to reduced numbers of activated CD4⁺ T cells in the lungs at this early time point. Furthermore, CD4⁺ T cells isolated from P. murina-infected TNFKO chimeras were unable to stimulate clearance of P. murina upon adoptive transfer to recombinase-deficient (RAG1KO) hosts. Together, these data indicate that B cell-derived TNF plays an important function in promoting CD4⁺ T cell expansion and production of TNF and facilitating protection against P. murina infection.     

Pre-treatment with IL-1β enhances the efficacy of MSC transplantation in DSS-induced colitis

Mesenchymal stem cells (MSCs) have been used experimentally for treating inflammatory disorders, partly due to their immunosuppressive properties. Although interleukin-1β (IL-1β) is one of the most important inflammatory mediators, growing evidence indicates that IL-1β signaling elicits the immunosuppressive properties of MSCs. However, it remains unclear how IL-1β signaling accomplishes this activity. Here, we focus on the therapeutic efficacy of IL-1β-primed MSCs in the dextran sulfate sodium (DSS)-induced colitis model, in addition to the underlining mechanisms. We first found that IL-1β-primed MSCs, without any observable phenotype change in vitro, significantly attenuated the development of DSS-induced murine colitis. Moreover, IL-1β-primed MSCs modulated the balance of immune cells in the spleen and the mesenteric lymph nodes (MLNs) through elevating cyclooxygenase-2 (COX-2), IL-6 and IL-8 expression and influencing the polarization of peritoneal macrophages. Importantly, IL-1β-primed MSCs possessed an enhanced ability to migrate to the inflammatory site of the gut via upregulation of chemokine receptor type 4 (CXCR4) expression. In summary, IL-1β-primed MSCs have improved efficacy in treating DSS-induced colitis, which at least partly depends on their increased immunosuppressive capacities and enhanced migration ability.     

Critical Roles of Inflammation and Apoptosis in Improved Survival in a Model of Hyperoxia-Induced Acute Lung Injury in Pneumocystis murina-Infected Mice

Pneumocystis infections increase host susceptibility to additional insults that would be tolerated in the absence of infection, such as hyperoxia. In an in vivo model using CD4-depleted mice, we previously demonstrated that Pneumocystis murina pneumonia causes significant mortality following an otherwise nonlethal hyperoxic insult. Infected mice demonstrated increased pulmonary inflammation and alveolar epithelial cell apoptosis compared to controls. To test the mechanisms underlying these observations, we examined expression of components of the Fas-Fas ligand pathway in P. murina-infected mice exposed to hyperoxia. Hyperoxia alone increased expression of Fas on the surface of type II alveolar epithelial cells; conversely, infection with P. murina led to increased lung expression of Fas ligand. We hypothesized that inhibition of inflammatory responses or direct inhibition of alveolar epithelial cell apoptosis would improve survival in P. murina-infected mice exposed to hyperoxia. Mice were depleted of CD4⁺ T cells and infected with P. murina and then were exposed to >95% oxygen for 4 days, followed by return to normoxia. Experimental groups received vehicle, dexamethasone, or granulocyte-macrophage colony-stimulating factor (GM-CSF). Compared with the vehicle-treated group, treatment with dexamethasone reduced Fas ligand expression and significantly improved survival. Similarly, treatment with GM-CSF, an agent we have shown protects alveolar epithelial cells against apoptosis, decreased Fas ligand expression and also improved survival. Our results suggest that the dual stresses of P. murina infection and hyperoxia induce lung injury via activation of the Fas-Fas ligand pathway and that corticosteroids and GM-CSF reduce mortality in P. murina-infected mice exposed to hyperoxic stress by inhibition of inflammation and apoptosis.Pneumocystis jirovecii infections remain a significant cause of morbidity and mortality in immunosuppressed individuals, including individuals with human immunodeficiency virus (HIV) infection and those receiving immunosuppressive therapy (27). Despite prompt institution of appropriate antibiotic therapy, a significant number of patients experience clinical deterioration and respiratory failure after hospital admission. The pathophysiologic mechanisms leading to deterioration in some patients have not been defined. Treatment with corticosteroids has become the standard of care for moderate to severe P. jirovecii pneumonia in patients with AIDS and has been found to decrease the rate of posthospitalization progression to respiratory failure (2, 7, 13, 22). Presumably, the beneficial effects of corticosteroids are mediated by blunting of the patients'' pulmonary inflammatory responses, but the mechanisms by which this improvement occurs are not understood.In previous work, we hypothesized that a secondary stress triggers respiratory failure in patients with P. jirovecii pneumonia (5). This secondary insult likely triggers an inflammatory response that results in detrimental effects to the host. Among potential triggers (acting alone or in combination) are drug effects, coincident bacterial infections, and supplemental oxygen therapy. We reasoned that oxidant stress might be a common mechanism precipitating progression to respiratory failure. To date, prospective or retrospective analysis of oxygen therapy as an independent predictor or respiratory failure has not been possible in patients with P. jirovecii pneumonia, with or without corticosteroid therapy. Therefore, we have used an animal model to test this concept. We previously developed a model in which mice were depleted of CD4⁺ T cells, infected with P. murina, and then exposed to a limited period of hyperoxia (5). This period of hyperoxia, by itself, caused minimal lung injury and no lethality. However, we found that P. murina-infected mice exposed to hyperoxia succumbed to this dual stress, whereas either individual insult alone did not result in death. We demonstrated that mice exposed to sublethal hyperoxia did not have an increased burden of organisms compared to mice maintained in normoxia. However, the dual stress of hyperoxia and P. murina infection resulted in significantly increased apoptosis of lung cells and in exuberant inflammatory responses. Histologically, we have identified increased apoptosis in alveolar macrophages and alveolar epithelial cells, but we have not examined the cells ex vivo or investigated the specific mechanisms by which this combined stress leads to alveolar epithelial cell apoptosis.Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a product of normal alveolar epithelial cells that has important effects on pulmonary innate immunity and surfactant homeostasis. We previously demonstrated that GM-CSF is critical for host defense against P. murina (25). GM-CSF is also an antiapoptotic factor for alveolar epithelial cells (24). In mice exposed to sustained hyperoxia, overexpression of GM-CSF conferred significant protection from alveolar epithelial cell injury and mortality (24). These effects were associated with reduced alveolar epithelial cell apoptosis, an effect that was reproduced with pharmacologic treatment with recombinant murine GM-CSF.In the present experiments, we hypothesized that the combination of inflammation due to P. murina infection and hyperoxic stress results in increased vulnerability of alveolar epithelial cells to Fas-mediated apoptosis, leading to lung injury and death. We determined that the dual insults of P. murina infection and hyperoxic stress induce components of the Fas-Fas ligand apoptotic pathway and found that treatment with GM-CSF improves survival in this immunologically relevant model of Pneumocystis pneumonia. Furthermore, treatment with corticosteroids improves survival in this model, recapitulating the benefit of treatment in human disease and providing a possible explanation for the benefits humans derive from treatment with an anti-inflammatory medication in the setting of acute infection.     

Novel Pneumocystis Antigen Discovery Using Fungal Surface Proteomics

Pneumonia due to the fungus Pneumocystis jirovecii is a life-threatening infection that occurs in immunocompromised patients. The inability to culture the organism as well as the lack of an annotated genome has hindered antigen discovery that could be useful in developing novel vaccine- or antibody-based therapies as well as diagnostics for this infection. Here we report a novel method of surface proteomics analysis of Pneumocystis murina that reliably detected putative surface proteins that are conserved in Pneumocystis jirovecii. This technique identified novel CD4⁺ T-cell epitopes as well as a novel B-cell epitope, Meu10, which encodes a glycosylphosphatidylinositol (GPI)-anchored protein thought to be involved in ascospore assembly. The described technique should facilitate the discovery of novel target proteins for diagnostics and therapeutics for Pneumocystis infection.     

Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.     

B Cells Modulate Systemic Responses to Pneumocystis murina Lung Infection and Protect On-Demand Hematopoiesis via T Cell-Independent Innate Mechanisms when Type I Interferon Signaling Is Absent

HIV infection results in a complex immunodeficiency due to loss of CD4⁺ T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag^−/−) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR^−/−) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag^−/− mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag^−/− mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4⁺ T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.     

Pneumocystis carinii exhibits a conserved meiotic control pathway

Pneumocystis carinii is an opportunistic fungus causing severe pneumonia in immune-compromised hosts. Recent evidence suggests that Pneumocystis exists as separate sex types, though definitive evidence is currently lacking. These studies were undertaken to determine whether Pneumocystis maintains functional meiotic control molecules, which are required for sexual life stages in eukaryotes. Using the Pneumocystis carinii Genome Project database, two partial sequences for meiotic control molecules were detected, namely, PCRan1, a presumptive meiotic control kinase, and PCMei2, a homologue to a primary activator of meiosis in Schizosaccharomyces pombe. Rapid amplification of cDNA ends was employed to obtain the full open reading frames and to further investigate the functions of these proteins. These presumptive meiotic control molecules were most homologous to molecules present in S. pombe (52% identical and 67% homologous for PCRan1 and 75% identical and 88% homologous for PCMei2 by BLAST analysis). Heterologous expression of these Pneumocystis meiotic genes in corresponding temperature-sensitive and knockout strains of S. pombe, respectively, further verified the functions of the PCRan1 and PCMei2 proteins. These proteins were further shown to control downstream components of the meiotic pathway in S. pombe. Lastly, in vitro kinase assays were used to determine that PCRan1p phosphorylates PCMei2p. These experiments represent the first characterization of any proteins in P. carinii involved in meiosis and indicate the presence of a conserved meiotic pathway in Pneumocystis. Elucidation of this pathway will be essential in gaining a greater understanding of this important opportunistic fungal pathogen.     

Cellular requirements for immunomodulatory effects caused by cell wall components of Paracoccidioides brasiliensis on antibody production

In a previous study, we reported an increase in the number of immunoglobulin-secreting cells and the augmentation of antibody production (IgM and IgG3) against unrelated antigens (sheep erythrocytes or bovine serum albumin (BSA)) in mice infected with the fungus Paracoccidioides brasiliensis as well as in mice inoculated with its cell wall preparation (CW). The immunomodulatory effect of the live fungus and CW preparation was dose-dependent and mainly restricted to the i.p. inoculation simultaneously to the BSA challenge by the i.v. route. In the present study, we investigated the active component of CW preparation upon the phenotype and also the degree of activation of possible target peritoneal cells involved in those phenomena. An insoluble polysaccharide fraction (F₁ fraction) mainly composed of β-glucan and chitin, and the purified β-glucan (BGPb) behaved as CW in the augmentation of early antibody production. The peritoneal mononuclear inflammatory cells induced by CW, F₁ fraction and BGPb were highly positive to α-naphthyl esterase staining; released low H₂O₂; expressed high levels of MHC-Ia^d molecules and produced inflammatory cytokines such as tumour necrosis factor-alpha (TNF-α) and IL-6. Phenotypic analysis by flow cytometry and immunohistochemical techniques of the inflammatory cells responding to F₁ fraction showed a prevalence of (CD11b/CD18, Mac-1)⁺ peritoneal macrophages. In addition, s.c. inoculation of F₁ fraction resulted in the formation of nodular, localized and not progressive granulomatous lesions with an accumulation of (CD11b/C18)⁺ macrophages. Adoptive transferred Mac-1 macrophages to immunized syngeneic recipient mice were able to cause an increase in anti-BSA antibody production. These results suggest that inflammatory (CD11b/CD18)⁺ macrophages may be related to immunological disturbances, caused by cell wall components of P. brasiliensis.