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.

Pneumocystis carinii is an opportunistic fungal pathogen that causes pneumonia (PCP) in immunocompromised individuals. Recent studies have demonstrated that the host's immune response is clearly responsible for the majority of the pathophysiological changes associated with PCP. P. carinii interacts closely with alveolar epithelial cells (AECs); however, the nature and pathological consequences of the epithelial response remain poorly defined. Monocyte chemotactic protein-1 (MCP-1) is involved in lung inflammation, immunity, and epithelial repair and is upregulated during PCP. To determine whether AECs are an important source of MCP-1 in the P. carinii-infected lung, in vivo and in vitro studies were performed. In situ hybridization showed that MCP-1 mRNA was localized to cells with morphological characteristics of AECs in the lungs of infected mice. In vitro studies demonstrated that P. carinii stimulated a time-and dose-dependent MCP-1 response in primary murine type II cells that was preceded by JNK activation. Pharmacological inhibition of JNK nearly abolished P. carinii-stimulated MCP-1 production, while ERK, p38 MAPK, and TNF receptor signaling were not required. Furthermore, delivery of a JNK inhibitory peptide specifically to pulmonary epithelial cells using a recombinant adenovirus vector blocked the early lung MCP-1 response following intratracheal instillation of infectious P. carinii. JNK inhibition did not affect P. carinii-stimulated production of macrophage inflammatory protein-2 in vitro or in vivo, indicating that multiple signaling pathways are activated in P. carinii-stimulated AECs. These data demonstrate that AECs respond to P. carinii in a proinflammatory manner that may contribute to the generation of immune-mediated lung injury. inflammation; epithelial; AIDS PNEUMONIA INDUCED by Pneumocystis carinii (PCP) continues to be the most common AIDS-defining illness as well as an important cause of morbidity and mortality in patients with a wide array of immunosuppressive conditions. Most recent studies indicate that, although the presence of P. carinii is obviously a factor in the development of lung injury, pulmonary inflammation is a major determinant of the severity of PCP (5,27,53,54). In AIDS patients with profound reductions in CD4 ϩ T cell numbers, bronchoalveolar lavage (BAL) fluid IL-8 and neutrophil concentrations, but not organism numbers, correlate with severity of PCP (5, 23, 28). In addition, clinical studies of immune-reconstituted PCP patients and controlled animal studies have both demonstrated that inflammatory mediators are released, and immune and inflammatory cells are recruited to the lung in response to P. carinii (2,25,38,39). More defined studies in mice have identified specific T cell subsets as having prominent roles in the lung injury associated with PCP (54, 57). For example, CD8 ϩ T cells are responsible for much of the PCP-associated lung injury that occurs in CD4-deficient hosts (17). Therefore, recent studies have focused on the mechanisms by which T cells accumu...

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-1β, 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.

.-Immune-mediated lung injury is an important component of Pneumocystis pneumonia (PcP)-related immunorestitution disease (IRD). However, the individual contribution of CD4 ϩ and CD8 ϩ T cells to the pathophysiology of IRD remains undetermined. Therefore, IRD was modeled in severe combined immunodeficient mice, and specific T cell depletion was used to determine how T cell subsets interact to affect the nature and severity of disease. CD4 ϩ cells were more abundant than CD8 ϩ cells during the acute stage of IRD that coincided with impaired pulmonary physiology and organism clearance. Conversely, CD8 ϩ cells were more abundant during the resolution phase following P. carinii clearance. Depletion of CD4 ϩ T cells protected mice from the acute pathophysiology of IRD. However, these mice could not clear the infection and developed severe PcP at later time points when a pathological CD8 ϩ T cell response was observed. In contrast, mice depleted of CD8 ϩ T cells efficiently cleared the infection but developed more severe disease, an increased frequency of IFN-␥-producing CD4 ϩ cells, and a prolonged CD4 ϩ T cell response than mice with both CD4 ϩ and CD8 ϩ cells. These data suggest that CD4 ϩ T cells mediate the acute respiratory disease associated with IRD. In contrast, CD8 ϩ T cells contributed to neither lung injury nor organism clearance when CD4 ϩ cells were present, but instead served to modulate CD4 function. In the absence of CD4 ϩ cells, CD8 ϩ T cells produced a nonprotective, pathological immune response. These data suggest that the interplay of CD4 ϩ and CD8 ϩ T cells affects the ultimate outcome of PcP-related IRD.inflammation; pulmonary physiology; acquired immune deficiency syndrome PNEUMOCYSTIS carinii IS an opportunistic fungus that causes pneumonia in immune-compromised patients (37), including patients with acquired immune deficiency syndrome (AIDS) (29), patients undergoing chemotherapy, and patients receiving treatment with immunosuppressive drugs during organ transplantation (34). Although exposure to P. carinii is nearly universal, as demonstrated by the appearance of anti-P. carinii antibody in 85% of children by the age of 20 mo (35), a period of immunosuppression is essential for the development of clinical disease (15,34). Several studies have demonstrated a positive correlation between the degree of inflammation and the severity of disease (4,23,36), suggesting that the pathogenesis of Pneumocystis pneumonia (PcP) is primarily inflammatory in nature, in that infection with P. carinii is necessary to cause pneumonia, but certain aspects of the immune response against P. carinii are responsible for the associated pathophysiology (30, 39). For example, AIDS patients who demonstrate a rapid recovery of CD4 ϩ T lymphocytes after institution of combined antiretroviral therapy may develop pulmonary decompensation in response to preexisting pulmonary infections, including P. carinii (26). This clinical syndrome has been termed "immunorestitution disease" (IRD) (9). At least one study that examined t...

While CD8؉ cells have been shown to contribute to lung injury during Pneumocystis carinii pneumonia (PCP), there are conflicting reports concerning the ability of CD8 ؉ cells to kill P. carinii. To address these two issues, we studied the effect of the presence of CD8 ؉ cells in two mouse models of PCP. In the reconstituted SCID mouse model, depletion of CD8؉ cells in addition to CD4 ؉ cells after reconstitution did not result in increased numbers of P. carinii cysts compared to the numbers of cysts in mice with only CD4 ؉ cells depleted. This result was observed regardless of whether the mice were reconstituted with naïve or P. carinii-sensitized lymphocytes. In contrast, reconstitution with sensitized lymphocytes resulted in more rapid onset of lung injury that was dependent on the presence of CD8؉ cells. The course of organism replication over a 6-week period was also examined in the CD4؉ -T-cell-depleted and CD4 ؉ -and CD8 ؉ -T-cell-depleted mouse model of PCP. Again, the organism burdens were identical at all times regardless of whether CD8؉ cells were present. Thus, in the absence of CD4 ؉ T cells, CD8 ؉ T cells are a key contributor to the inflammatory lung injury associated with PCP. However, we were unable to demonstrate an in vivo effect of these cells on the course of P. carinii infection. Efficient control of Pneumocystis carinii infection requires normally functioning CD4ϩ T cells (9, 12). Although CD8 ϩ T cells have been reported to be able to produce a modest decrease in the level of P. carinii infection in the absence of CD4 ϩ T cells (2), the mechanism for this effect is not obvious since P. carinii is, as far as we know, an extracellular pathogen. Our observations have failed to demonstrate any killing of P. carinii by CD8 ϩ T cells (16; unpublished observations). On the other hand, there is clear evidence that CD8 ϩ T cells make a biologically significant contribution to the immune-mediated inflammatory insult to the lung during P. carinii pneumonia (PCP) (16,17). Therefore, defining the contribution of CD8 ϩ T cells to the control of infection compared to the exacerbation of lung injury is important in developing improved adjunctive therapies for PCP targeted at suppressing CD8 ϩ T cells. To more precisely define the role of CD8 ϩ T cells in the control of P. carinii f. sp. muris, we carried out a series of experiments with two mouse models of PCP. Using the immune reconstituted SCID mouse model, we hypothesized that if CD8 ϩ T cells can in fact kill P. carinii, then exposure (i.e., sensitization) of CD8 ϩ T cells to P. carinii by immunization before they are infused into P. carinii-infected SCID mice should result in enhanced clearance of organisms. Using donor splenocytes from P. carinii-immunized mice also allowed us to further examine the effect of CD8 ϩ T cells on immune-mediated inflammatory injury during PCP. With regard to the latter point, we hypothesized that if CD8 ϩ T cells play a key role in initiating the inflammatory response during PCP, then CD8 ϩ T cells from immunized donor ...