IFN-γ Limits Immunopathogenesis but Delays Fungal Clearance during Pneumocystis Pneumonia.

High levels of IFN-γ are produced in the lung during an adaptive immune response to Pneumocystis, but the effects of this prototypical Th1 cytokine on fungal clearance and immunopathogenesis have not been fully defined. Therefore, Pneumocystis-infected immunodeficient mice were immune reconstituted and administered control or anti-IFN-γ neutralizing Ab to determine how IFN-γ regulates the balance between host defense and immune-mediated lung injury. Mice treated with anti-IFN-γ demonstrated an initial worsening of Pneumocystis pneumonia-related immunopathogenesis, with greater weight loss, heightened lung inflammation, and more severe pulmonary function deficits than control mice. However, IFN-γ neutralization also enhanced macrophage phagocytosis of Pneumocystis and accelerated fungal clearance. When anti-IFN-γ-treated mice were also given IL-4 and IL-13 to promote a Th2-biased lung environment, the accelerated fungal clearance was preserved, but the severity of immunopathogenesis was reduced, and a more rapid recovery was observed. A direct suppressive effect of IFN-γ on macrophages was required but was not solely responsible for delayed fungal clearance, suggesting that IFN-γ acts through multiple mechanisms that likely include modulation of both macrophage and Th polarization. Enhanced Pneumocystis clearance in anti-IFN-γ-treated and IFN-γR-deficient mice was associated with significantly elevated IL-17+ CD4+ T cells and IL-17 protein in the lungs. Furthermore, neutralization of IL-17, but not IL-4, signaling blocked the accelerated fungal clearance observed in anti-IFN-γ-treated mice. Together, these data demonstrate that although IFN-γ delays fungal clearance by suppressing the lung Th17 response, it also serves an important regulatory role that limits immunopathogenesis and preserves pulmonary function.

The Dual Benefit of Sulfasalazine on Pneumocystis Pneumonia-Related Immunopathogenesis and Antifungal Host Defense Does Not Require IL-4Rα-Dependent Macrophage Polarization.

Pneumocystis is a respiratory fungal pathogen that is among the most frequent causes of life-threatening pneumonia (PcP) in immunocompromised hosts. Alveolar macrophages play an important role in host defense against Pneumocystis, and several studies have suggested that M2 polarized macrophages have anti-Pneumocystis effector activity. Our prior work found that the immunomodulatory drug sulfasalazine (SSZ) provides a dual benefit during PcP-related immune reconstitution inflammatory syndrome (IRIS) by concurrently suppressing immunopathogenesis while also accelerating macrophage-mediated fungal clearance. The benefits of SSZ were associated with heightened Th2 cytokine production and M2 macrophage polarization. Therefore, to determine whether SSZ improves the outcome of PcP through a mechanism that requires Th2-dependent M2 polarization, RAG2-/- mice lacking interleukin 4 receptor alpha chain (IL-4Rα) on macrophage lineage cells were generated. As expected, SSZ treatment dramatically reduced the severity of PcP-related immunopathogenesis and accelerated fungal clearance in immune-reconstituted RAG2-/- mice. Similarly, SSZ treatment was also highly effective in immune-reconstituted RAG2/IL-4Rα-/- and RAG2/gamma interferon receptor (IFN-γR)-/- mice, demonstrating that neither IL-4Rα-dependent M2 nor IFN-γR-dependent M1 macrophage polarization programs were required for the beneficial effects of SSZ. Despite the fact that macrophages from RAG2/IL-4Rα-/- mice could not respond to the Th2 cytokines IL-4 and IL-13, M2-biased alveolar macrophages were identified in the lungs following SSZ treatment. These data demonstrate that not only does SSZ enhance phagocytosis and fungal clearance in the absence of macrophage IL-4Rα signaling, but also that SSZ promotes M2 macrophage polarization in an IL-4Rα-independent manner. These findings could have implications for the treatment of PcP and other diseases in which M2 polarization is beneficial.

Combination Immunotherapy with Passive Antibody and Sulfasalazine Accelerates Fungal Clearance and Promotes the Resolution of Pneumocystis-Associated Immunopathogenesis.

The pulmonary immune response protects healthy individuals against Pneumocystis pneumonia (PcP). However, the immune response also drives immunopathogenesis in patients who develop severe PcP, and it is generally accepted that optimal treatment requires combination strategies that promote fungal killing and also provide effective immunomodulation. The anti-inflammatory drug sulfasalazine programs macrophages for enhanced Pneumocystis phagocytosis and also suppresses PcP-related immunopathogenesis. Anti-Pneumocystis antibody opsonizes Pneumocystis organisms for greater phagocytosis and may also mask antigens that drive immunopathogenesis. Thus, we hypothesized that combining antibody and sulfasalazine would have the dual benefit of enhancing fungal clearance while dampening immunopathogenesis and allow the rescue of severe PcP. To model a clinically relevant treatment scenario in mice, therapeutic interventions were withheld until clear symptoms of pneumonia were evident. When administered individually, both passive antibody and sulfasalazine improved pulmonary function and enhanced Pneumocystis clearance to similar degrees. However, combination treatment with antibody and sulfasalazine produced a more rapid improvement, with recovery of body weight, a dramatic improvement in pulmonary function, reduced lung inflammation, and the rapid clearance of the Pneumocystis organisms. Accelerated fungal clearance in the combination treatment group was associated with a significant increase in macrophage phagocytosis of Pneumocystis Both passive antibody and sulfasalazine resulted in the suppression of Th1 cytokines and a marked increase in lung macrophages displaying an alternatively activated phenotype, which were enhanced by combination treatment. Our data support the concept that passive antibody and sulfasalazine could be an effective and specific adjunctive therapy for PcP, with the potential to accelerate fungal clearance while attenuating PcP-associated immunopathogenesis.

Immunization with Pneumocystis Cross-Reactive Antigen 1 (Pca1) Protects Mice against Pneumocystis Pneumonia and Generates Antibody to Pneumocystis jirovecii.

Pneumocystis pneumonia (PcP) is a life-threatening infection that affects immunocompromised individuals. Nearly half of all PcP cases occur in those prescribed effective chemoprophylaxis, suggesting that additional preventive methods are needed. To this end, we have identified a unique mouse Pneumocystis surface protein, designated Pneumocystis cross-reactive antigen 1 (Pca1), as a potential vaccine candidate. Mice were immunized with a recombinant fusion protein containing Pca1. Subsequently, CD4+ T cells were depleted, and the mice were exposed to Pneumocystis murina Pca1 immunization completely protected nearly all mice, similar to immunization with whole Pneumocystis organisms. In contrast, all immunized negative-control mice developed PcP. Unexpectedly, Pca1 immunization generated cross-reactive antibody that recognized Pneumocystis jirovecii and Pneumocystis carinii Potential orthologs of Pca1 have been identified in P. jirovecii Such cross-reactivity is rare, and our findings suggest that Pca1 is a conserved antigen and potential vaccine target. The evaluation of Pca1-elicited antibodies in the prevention of PcP in humans deserves further investigation.

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.

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.

Selective ablation of lung epithelial IKK2 impairs pulmonary Th17 responses and delays the clearance of Pneumocystis.

Pneumocystis is an atypical fungal pathogen that causes severe, often fatal pneumonia in immunocompromised patients. Healthy humans and animals also encounter this pathogen, but they generate a protective CD4(+) T cell-dependent immune response that clears the pathogen with little evidence of disease. Pneumocystis organisms attach tightly to respiratory epithelial cells, and in vitro studies have demonstrated that this interaction triggers NF-κB-dependent epithelial cell responses. However, the contribution of respiratory epithelial cells to the normal host response to Pneumocystis remains unknown. IκB kinase 2 (IKK2) is the upstream kinase that is critical for inducible NF-κB activation. To determine whether IKK2-dependent lung epithelial cell (LEC) responses contribute to the anti-Pneumocystis immune response in vivo, transgenic mice with LEC-specific deletion of IKK2 (IKK2(ΔLEC)) were generated. Compared to wild-type mice, IKK2(ΔLEC) mice exhibited a delayed onset of Th17 and B cell responses in the lung and delayed fungal clearance. Importantly, delayed Pneumocystis clearance in IKK2(ΔLEC) mice was associated with an exacerbated immune response, impaired pulmonary function, and altered lung histology. These data demonstrate that IKK2-dependent LEC responses are important regulators of pulmonary adaptive immune responses and are required for optimal host defense against Pneumocystis infection. LECs likely set the threshold for initiation of the pulmonary immune response and serve to prevent exacerbated lung inflammation by promoting the rapid control of respiratory fungal infection.

The alveolar epithelial cell chemokine response to pneumocystis requires adaptor molecule MyD88 and interleukin-1 receptor but not toll-like receptor 2 or 4.

Pneumocystis is an opportunistic fungal pathogen that causes pneumonia in a variety of clinical settings. An early step in Pneumocystis infection involves the attachment of organisms to alveolar epithelial cells (AECs). AECs produce chemokines in response to Pneumocystis stimulation, but the upstream host-pathogen interactions that activate AEC signaling cascades are not well-defined. MyD88 is an adaptor molecule required for activation of proinflammatory signaling cascades following Toll-like receptor (TLR)-dependent recognition of conserved molecular patterns on pathogens. To determine whether the TLR/MyD88 pathway is required for the AEC chemokine response to Pneumocystis, wild-type (WT) and MyD88-deficient AECs were incubated with Pneumocystis. As expected, WT AECs produced CCL2 and CXCL2 following Pneumocystis stimulation. In contrast, MyD88-deficient AECs were severely impaired in their ability to respond to Pneumocystis. MyD88-deficient AECs did not display Pneumocystis-induced Jun N-terminal protein kinase activation and produced much less chemokine than Pneumocystis-stimulated WT AECs. Using a panel of TLR agonists, primary murine AECs were found to respond vigorously to TLR2 and TLR4 agonists. However, the AEC chemokine response to Pneumocystis did not require TLR2 or TLR4. Surprisingly, the interleukin-1 receptor (IL-1R) was required for an AEC chemokine response to Pneumocystis. The role of MyD88 in early responses during Pneumocystis infection was supported by in vivo studies demonstrating that MyD88-deficient mice showed impaired Pneumocystis-stimulated chemokine production and impaired inflammatory cell recruitment. These data indicate an important role for MyD88 in the AEC inflammatory response to Pneumocystis.

Immune modulation with sulfasalazine attenuates immunopathogenesis but enhances macrophage-mediated fungal clearance during Pneumocystis pneumonia.

Although T cells are critical for host defense against respiratory fungal infections, they also contribute to the immunopathogenesis of Pneumocystis pneumonia (PcP). However, the precise downstream effector mechanisms by which T cells mediate these diverse processes are undefined. In the current study the effects of immune modulation with sulfasalazine were evaluated in a mouse model of PcP-related Immune Reconstitution Inflammatory Syndrome (PcP-IRIS). Recovery of T cell-mediated immunity in Pneumocystis-infected immunodeficient mice restored host defense, but also initiated the marked pulmonary inflammation and severe pulmonary function deficits characteristic of IRIS. Sulfasalazine produced a profound attenuation of IRIS, with the unexpected consequence of accelerated fungal clearance. To determine whether macrophage phagocytosis is an effector mechanism of T cell-mediated Pneumocystis clearance and whether sulfasalazine enhances clearance by altering alveolar macrophage phagocytic activity, a novel multispectral imaging flow cytometer-based method was developed to quantify the phagocytosis of Pneumocystis in vivo. Following immune reconstitution, alveolar macrophages from PcP-IRIS mice exhibited a dramatic increase in their ability to actively phagocytose Pneumocystis. Increased phagocytosis correlated temporally with fungal clearance, and required the presence of CD4(+) T cells. Sulfasalazine accelerated the onset of the CD4(+) T cell-dependent alveolar macrophage phagocytic response in PcP-IRIS mice, resulting in enhanced fungal clearance. Furthermore, sulfasalazine promoted a TH2-polarized cytokine environment in the lung, and sulfasalazine-enhanced phagocytosis of Pneumocystis was associated with an alternatively activated alveolar macrophage phenotype. These results provide evidence that macrophage phagocytosis is an important in vivo effector mechanism for T cell-mediated Pneumocystis clearance, and that macrophage phenotype can be altered to enhance phagocytosis without exacerbating inflammation. Immune modulation can diminish pulmonary inflammation while preserving host defense, and has therapeutic potential for the treatment of PcP-related immunopathogenesis.

Ablation of TNF-RI/RII expression in Alzheimer's disease mice leads to an unexpected enhancement of pathology: implications for chronic pan-TNF-α suppressive therapeutic strategies in the brain.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by severe memory loss and cognitive impairment. Neuroinflammation, including the extensive production of pro-inflammatory molecules and the activation of microglia, has been implicated in the disease process. Tumor necrosis factor (TNF)-α, a prototypic pro-inflammatory cytokine, is elevated in AD, is neurotoxic, and colocalizes with amyloid plaques in AD animal models and human brains. We previously demonstrated that the expression of TNF-α is increased in AD mice at ages preceding the development of hallmark amyloid and tau pathological features and that long-term expression of this cytokine in these mice leads to marked neuronal death. Such observations suggest that TNF-α signaling promotes AD pathogenesis and that therapeutics suppressing this cytokine's activity may be beneficial. To dissect TNF-α receptor signaling requirements in AD, we generated triple-transgenic AD mice (3xTg-AD) lacking both TNF-α receptor 1 (TNF-RI) and 2 (TNF-RII), 3xTg-ADxTNF-RI/RII knock out, the cognate receptors of TNF-α. These mice exhibit enhanced amyloid and tau-related pathological features by the age of 15 months, in stark contrast to age-matched 3xTg-AD counterparts. Moreover, 3xTg-ADxTNF-RI/RII knock out-derived primary microglia reveal reduced amyloid-ß phagocytic marker expression and phagocytosis activity, indicating that intact TNF-α receptor signaling is critical for microglial-mediated uptake of extracellular amyloid-ß peptide pools. Overall, our results demonstrate that globally ablated TNF receptor signaling exacerbates pathogenesis and argues against long-term use of pan-anti-TNF-α inhibitors for the treatment of AD.

Anti-CD3 antibody decreases inflammation and improves outcome in a murine model of Pneumocystis pneumonia.

The T cell-mediated immune response elicited by Pneumocystis plays a key role in pulmonary damage and dysfunction during Pneumocystis carinii pneumonia (PcP). Mice depleted of CD4(+) and CD8(+) T cells prior to infection are markedly protected from PcP-related respiratory deficit and death, despite progressive lung infection. However, the therapeutic effectiveness of Ab-mediated disruption of T cell function in mice already displaying clinical symptoms of disease has not been determined. Therefore, a murine model of PcP-related immune reconstitution inflammatory syndrome was used to assess whether Ab to the pan-T cell molecule CD3 is effective for reducing the severity of PcP when administered after the onset of disease. Mice that received anti-CD3 Ab exhibited a rapid and dramatic halt in the PcP-associated pulmonary function decline within 1 week after treatment, and a striking enhancement of survival rate compared with mice receiving the control Ab. Physiologic improvement in anti-CD3 treated mice was associated with a significant reduction in the number of CD4(+) and CD8(+) T cells recovered in lung lavage fluid. This effectiveness of anti-CD3 was noted whether the mice also received antibiotic therapy with trimethoprim-sulfamethoxazole. These data suggest that monoclonal Ab-mediated disruption of T cell function may represent a specific and effective adjunctive therapy to rapidly reverse the ongoing pathologic immune response occurring during active PcP. Thus, the anti-human CD3 monoclonal Ab OKT3, which is already in clinical use, has the potential to be developed as an adjunctive therapy for PcP.

Parenchymal cell TNF receptors contribute to inflammatory cell recruitment and respiratory failure in Pneumocystis carinii-induced pneumonia.

The opportunistic organism Pneumocystis carinii (Pc) produces a life-threatening pneumonia (PcP) in patients with low CD4(+) T cell counts. Animal models of HIV-AIDS-related PcP indicate that development of severe disease is dependent on the presence of CD8(+) T cells and the TNF receptors (TNFR) TNFRsf1a and TNFRsf1b. To distinguish roles of parenchymal and hematopoietic cell TNF signaling in PcP-related lung injury, murine bone marrow transplant chimeras of wild-type, C57BL6/J, and TNFRsf1a/1b double-null origin were generated, CD4(+) T cell depleted, and inoculated with Pc. As expected, C57 --> C57 chimeras (donor marrow --> recipient) developed significant disease as assessed by weight loss, impaired pulmonary function (lung resistance and dynamic lung compliance), and inflammatory cell infiltration. In contrast, TNFRsf1a/1b(-/-) --> TNFRsf1a/1b(-/-) mice were relatively mildly affected despite carrying the greatest organism burden. Mice solely lacking parenchymal TNFRs (C57 --> TNFRsf1a/1b(-/-)) had milder disease than did C57 --> C57 mice. Both groups of mice with TNFR-deficient parenchymal cells had low bronchoalveolar lavage fluid total cell counts and fewer lavageable CD8(+) T cells than did C57 --> C57 mice, suggesting that parenchymal TNFR signaling contributes to PcP-related immunopathology through the recruitment of damaging immune cells. Interestingly, mice with wild-type parenchymal cells but TNFRsf1a/1b(-/-) hematopoietic cells (TNFRsf1a/1b(-/-) --> C57) displayed exacerbated disease characterized by increased MCP-1 and KC production in the lung and increased macrophage and lymphocyte numbers in the lavage, indicating a dysregulated immune response. This study supports a key role of parenchymal cell TNFRs in lung injury induced by Pc and a potential protective effect of receptors on radiosensitive, bone marrow-derived cells.

Murine mechanical ventilation stimulates alveolar epithelial cell proliferation.

High tidal volume mechanical ventilation can cause inflammation and lung damage. Mechanical strain is also necessary for normal lung growth. The current work was performed to determine if mechanical ventilation with clinically utilized tidal volumes stimulates a proliferative response in the lung. Six- to 8-week-old C57/Bl6 mice, anesthetized with ketamine/xylozine, were ventilated for 6 hours with 10 mL/kg tidal volume, positive end-expiratory pressure (PEEP) 3cm H(2)O. Pulmonary function testing demonstrated decreased compliance within 3 hours of ventilation. Assessment of bronchoalveolar lavage (BAL) demonstrated no significant increase in lactate dehydrogenase, total lavagable cell number, or total protein after ventilation. There was evidence of inflammation in the lungs of ventilated mice, with an increased percentage of lymphocytes and neutrophils in BAL, and an increase in macrophage inflammatory protein (MIP)-2 and interleukin (IL)-1beta message in lung tissue. Immunohistochemistry of inflation-fixed lungs demonstrated increased alveolar cell proliferation, as measured by both proliferating cell nuclear antigen and Ki67 staining. Dual staining confirmed that proliferating cells labeled with proSP-B, demonstrating that ventilation induces proliferation of alveolar type II cells. Ventilation did not increase apoptosis in alveolar type II cells, as measured by TUNEL staining. Ventilation at low tidal volumes leads to a mild inflammatory response and alveolar epithelial cell proliferation.

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.

CD73 regulates anti-inflammatory signaling between apoptotic cells and endotoxin-conditioned tissue macrophages.

The phagocytosis of apoptotic cells (efferocytosis) shifts macrophages to an anti-inflammatory state through a set of still poorly understood soluble and cell-bound signals. Apoptosis is a common feature of inflamed tissues, and efferocytosis by tissue macrophages is thought to promote the resolution of inflammation. However, it is not clear how the exposure of tissue macrophages to inflammatory cues (e.g., PAMPs, DAMPs) in the early stages of inflammation affects immune outcomes of macrophage-apoptotic cell interactions occurring at later stages of inflammation. To address this, we used low-dose endotoxin conditioning (LEC, 1 ng/ml LPS 18 h) of mouse resident peritoneal macrophages (RPMФ) to model the effects of suboptimal (i.e., non-tolerizing), antecedent TLR activation on macrophage inflammatory responses to apoptotic cells. Compared with unconditioned macrophages (MФ), LEC-MФ showed a significant enhancement of apoptotic cell-driven suppression of many inflammatory cytokines (e.g., TNF, MIP-1ß, MCP-1). We then found that enzymatic depletion of adenosine or inhibition of the adenosine receptor A2a on LEC-MФ abrogated apoptotic cell suppression of TNF, and this suppression was entirely dependent on the ecto-enzyme CD73 (AMPadenosine) but not CD39 (ATPAMP), both of which are highly expressed on RPMФ. In addition to a requirement for CD73, we also show that Adora2a levels in macrophages are a critical determinant of TNF suppression by apoptotic cells. LEC treatment of RPMФ led to a ~3-fold increase in Adora2a and a ~28-fold increase in adenosine sensitivity. Moreover, in RAW264.7 cells, ectopic expression of both A2a and CD73 was required for TNF suppression by apoptotic cells. In mice, mild, TLR4-dependent inflammation in the lungs and peritoneum caused a rapid increase in macrophage Adora2a and Adora2b levels, and CD73 was required to limit neutrophil influx in this peritonitis model. Thus immune signaling via the CD73-A2a axis in macrophages links early inflammatory events to subsequent immune responses to apoptotic cells.

Immunopathogenesis of Pneumocystis carinii pneumonia.

Pneumocystis carinii pneumonia (PCP) is a life-threatening infection that occurs in immunocompromised individuals, particularly those with advanced human immunodeficiency virus (HIV) infection. Interestingly, morbidity and mortality is related to the underlying cause of immunosuppression, with AIDS patients faring better than oncology patients for example. In addition, the prognosis of PCP has been correlated with markers of inflammation rather than with organism numbers. There is now increasing evidence that lung damage occurring during PCP is a result of the type and extent of the host inflammatory response to P. carinii rather than a result of direct damage by the organism. This review will discuss the experimental and clinical data demonstrating how the host-mediated inflammatory response to infection with P. carinii determines the ultimate outcome of PCP. A better understanding of the pathophysiology of PCP should lead to the development of improved therapies for the treatment of PCP.

The triterpenoid CDDO-Me inhibits bleomycin-induced lung inflammation and fibrosis.

Pulmonary Fibrosis (PF) is a devastating progressive disease in which normal lung structure and function is compromised by scarring. Lung fibrosis can be caused by thoracic radiation, injury from chemotherapy and systemic diseases such as rheumatoid arthritis that involve inflammatory responses. CDDO-Me (Methyl 2-cyano-3,12-dioxooleana-1,9(11)dien-28-oate, Bardoxolone methyl) is a novel triterpenoid with anti-fibrotic and anti-inflammatory properties as shown by our in vitro studies. Based on this evidence, we hypothesized that CDDO-Me would reduce lung inflammation, fibrosis and lung function impairment in a bleomycin model of lung injury and fibrosis. To test this hypothesis, mice received bleomycin via oropharyngeal aspiration (OA) on day zero and CDDO-Me during the inflammatory phase from days -1 to 9 every other day. Bronchoalveolar lavage fluid (BALF) and lung tissue were harvested on day 7 to evaluate inflammation, while fibrosis and lung function were evaluated on day 21. On day 7, CDDO-Me reduced total BALF protein by 50%, alveolar macrophage infiltration by 40%, neutrophil infiltration by 90% (p≤0.01), inhibited production of the inflammatory cytokines KC and IL-6 by over 90% (p≤0.001), and excess production of the pro-fibrotic cytokine TGFß by 50%. CDDO-Me also inhibited α-smooth muscle actin and fibronectin mRNA by 50% (p≤0.05). On day 21, CDDO-Me treatment reduced histological fibrosis, collagen deposition and αSMA production. Lung function was significantly improved at day 21 by treatment with CDDO-Me, as demonstrated by respiratory rate and dynamic compliance. These new findings reveal that CDDO-Me exhibits potent anti-fibrotic and anti-inflammatory properties in vivo. CDDO-Me is a potential new class of drugs to arrest inflammation and ameliorate fibrosis in patients who are predisposed to lung injury and fibrosis incited by cancer treatments (e.g. chemotherapy and radiation) and by systemic autoimmune diseases.

Immune Modulation as Adjunctive Therapy for Pneumocystis pneumonia.

Pneumocystis is an opportunistic fungal respiratory pathogen that causes life-threatening pneumonia (Pcp) in patients suffering from defects in cell-mediated immunity, including those with acquired immunodeficiency syndrome (AIDS) and immunosuppression secondary to chemotherapy or organ transplantation. Despite major advances in health care, the mortality associated with Pcp has changed little over the past 25 years. Pcp remains a leading cause of death among HIV infected patients, with mortality rates of 50% or higher for patients developing severe Pcp. In addition, as more potent immunosuppressive therapies are developed for chronic inflammatory diseases, more cases of Pcp are occurring in non-HIV patients and in previously unreported clinical settings. These features highlight the importance of developing a better understanding of the pathogenesis of this disease, and the need to search for new therapeutic strategies to improve the outcome of Pcp patients. Immune-mediated inflammatory responses play an important role in the pathogenesis of Pcp, and may be even more significant in determining the outcome of Pcp than direct damage due to the organism itself. In this review we will summarize the immunopathogenic mechanisms that contribute to Pcp-associated lung injury, and discuss the potential to target these pathways for adjunctive immune modulation therapy for Pcp.

Mixed Lineage Kinase 3 deficiency delays viral clearance in the lung and is associated with diminished influenza-induced cytopathic effect in infected cells.

Influenza virus leads to acute respiratory disease resulting in seasonal epidemics and periodic pandemics. Little is known about the signaling events that regulate host defense to influenza. One particular pathway, the c-Jun amino-terminal kinase (JNK) cascade is activated following influenza infection and blocking JNK leads to enhanced viral replication. We hypothesize that Mixed Lineage Kinase 3 (MLK3), an upstream regulator of JNK, is involved in the host response to influenza. To test this, wild-type and MLK3-/- mice were infected with pathogenic strain of influenza A virus, A/PR/8/34 (PR8). Although, cellular and humoral immune responses were similar between wild-type and MLK3-/- hosts, the viral load in the lungs was comparatively higher in MLK3-/- mice at day 8 post-infection. Consistent with this, MLK3-/- murine lung fibroblast and epithelial cells had prolonged survival and increased virion production following infection compared to wild-type. These findings support a role for MLK3 in viral production during influenza infection.