IRIS is a clinical manifestation of PcP that occurs in certain patients when cell-mediated immunity is restored following a period of immune suppression and infection 
. The resulting acute pulmonary inflammatory response restores host defense, but also produces severe pathology. The current study demonstrates that modulation of the immune response dramatically reduces the severity of PcP-related IRIS. The SSZ-mediated reduction in physiological impairment was associated with abrogated cytokine responses, reduced immune cell recruitment to the lung, and reduced histological evidence of inflammation. Unexpectedly, SSZ did not impair, but actually enhanced the host's ability to clear the Pc infection, indicating that the immune pathways leading to injury are at least partly independent of the pathways leading to clearance. Macrophages are equipped to recognize and eliminate pathogens as well as promote or resolve inflammation. To test the hypothesis that SSZ enhances Pc clearance via downstream functional alteration of AMs, a multispectral imaging flow cytometry-based method was developed to assess and quantify the in vivo
Pc phagocytic activity of AM. This technology demonstrated that AM are effector cells for CD4+
T-cell mediated Pc clearance, and that SSZ enhances clearance by accelerating the AM phagocytic response. Subsequent studies found that SSZ promotes alternative activation of AMs, which is associated with reduced immunopathogenesis, but enhanced phagocytosis and fungal clearance. These data demonstrate that AM can be phenotypically modified to enhance fungal phagocytosis and clearance without ehnancing their pro-inflammatory properties, and also provide in vivo
evidence that macrophage phagocytosis is the mechanism of CD4+
T cell-dependent Pc clearance from the lung.
SSZ is a clinically important immunomodulatory therapy for inflammatory diseases such as inflammatory bowel disease and rheumatoid arthritis 
. Most of the beneficial effects of SSZ are attributed to its function as a potent inhibitor of NF-κB. SSZ directly inhibits the activity of Inhibitor of κB Kinase (IKK), effectively preventing downstream κB-dependent transcriptional events 
. Recent clinical studies have confirmed that the beneficial effects of SSZ in patients with ulcerative colitis are in fact related to inhibition of NF-κB activation in the mucosa, which results in reduced cytokine production, and less severe inflammation 
. In addition to IKK inhibition, other mechanisms of SSZ action have been described. SSZ inhibits 5-aminoimidazole-4-carboxamidoribonucleotide transformylase causing the release of adenosine 
, which controls inflammation at least partly through inhibition of NF-κB signaling 
. Other investigators found that SSZ may alleviate inflammation in a mouse model of inflammatory bowel disease by interacting with PPAR (peroxisome proliferator-activated receptor) nuclear receptors 
. It is noteworthy that a common mechanism of all of these interactions is related to NF-κB inhibition, and it seems likely that SSZ-mediated blockade of NF-κB is central to the beneficial effects observed in our model. In fact, a highly specific IKK inhibitor, BMS-345541, mimicked the beneficial effects of SSZ on PcP-related lung injury and pulmonary dysfunction, suggesting that NF-κB plays an important role in the immune cascade leading to the development of PcP. However, BMS-345541 did not enhance pathogen clearance. Therefore, SSZ may have other, IKK-independent, immunomodulatory properties that account for the beneficial effects on AM phagocytosis and pathogen clearance.
The beneficial action of SSZ may result from its effects on a single cell type, or more likely, from its combined effects on several cell types that contribute to injury and disease. Potential lymphocyte targets of SSZ include CD4+
, and B lymphocytes. SSZ has pro-apoptotic effects on activated T cells 
, which may contribute to the reduced T cell numbers and inflammation found in PcP-related IRIS. SSZ also influences macrophage function through the induction of apoptosis, as well as alteration of macrophage inflammatory responses 
. SSZ blocked TNF production and also abrogated IL-12 expression and NO production by stimulated macrophages 
. Modification of macrophage IL-12 may represent a mechanism by which SSZ alters the nature of the T cell response during IRIS. NF-κB is also involved in pulmonary epithelial cell inflammatory responses to Pc 
, providing another potential target for the action of SSZ. While the immunopathology associated with PcP and IRIS requires T cells, other cell types likely contribute to the overall disease process, and therefore the effectiveness of SSZ reported here likely results from multiple points of action.
Our studies have found that SSZ produces a TH2 shift in the lung cytokine environment during PcP-related IRIS, and that this shift is reflected in the phenotype of alveolar macrophages. TH2 cytokines lead to alternative activation of macrophages, and consistent with a TH2 cytokine shift we found that AMs isolated from SSZ-treated mice express high levels of the AAM marker ARG, but low levels of the CAM marker INOS (). In contrast, AMs from PBS-treated IRIS mice display a CAM phenotype with high expression of INOS. It is notable that despite a well-documented role for INOS in host defense, these data suggest that enhanced Pc phagocytosis in SSZ-treated mice is associated with an alternatively activated AM phenotype with low expression of INOS. Based on our results, we believe that increased phagocytosis by alternatively activated macrophages is the mechanism of enhanced Pc clearance. However, a role for INOS in Pc killing cannot be excluded. Although we observe less staining in AMs from SSZ-treated mice, they are not totally devoid of INOS protein. More extensive studies will be required to determine the contribution of INOS in this model.
Although we have not demonstrated that the TH2 shift is solely responsible for the beneficial effects of SSZ during PcP, it is possible that TH2 cytokines acting through AAM effectors can increase fungal clearance while reducing immunopathogenesis. For example, TH2 cytokines enhance macrophage phagocytosis of Candida albicans
by inducing macrophage expression of mannose receptor (MR) 
and dectin-1 
. These pattern recognition molecules are markers of the AAM phenotype, and have known roles in anti-fungal host defense. Similarly a TH2 shift in SSZ-treated mice could enhance phagocytosis of Pc by eliciting AAM with increased expression of MR and dectin-1. In addition, a TH2 shift may also attenuate the immunopathogenesis of PcP by reducing the production of pro-inflammatory TH1 cytokines, while enhancing production of anti-inflammatory TH2 cytokines. Elevated lung levels of the TH1 cytokines TNF-α and IFN-γ are associated with PcP-related lung injury and respiratory impairment 
. In contrast, TH2-derived AAMs produce the potent anti-inflammatory cytokines IL-10 and TGF- β 
, which can dampen inflammatory responses and may contribute to the reduced inflammation and injury in SSZ-treated mice. Importantly, the anti-inflammatory potential of AAMs has been established in vivo
by studies showing that the adoptive transfer of in vitro
programmed AAMs attenuates immunopathogenesis in mouse models of inflammatory disease 
. Although these findings are consistent with a SSZ-induced shift in the polarity of the T cell response, further studies are required to establish whether TH2 cytokines and alternative activation of AMs are directly responsible for the beneficial effects of SSZ during PcP.
Clinical studies have found that the severity of PcP correlates with the degree of pulmonary inflammation, but not with organism burden 
. Controlled animal studies support these clinical observations, and have provided direct evidence that the immune response is a major pathogenic component of PcP 
. Consequently, antibiotic treatment does not always produce rapid improvement of patients with severe PcP, because organisms and antigen may continue to drive the pathological immune response. The efficacy of SSZ in dramatically attenuating the severity of PcP supports the contention that adjunctive immunomodulatory therapy that target the T cell response is critical to optimal treatment of patients. Currently, adjunctive corticosteroids are commonly used for the clinical treatment of PcP. The broad anti-inflammatory and immunosuppressive properties of steroids are presumed to provide benefit, but concrete evidence that steroids improve survival is lacking. Our group has recently published a study demonstrating that specific disruption of the T cell response to Pc with anti-CD3 antibody has beneficial effects in a mouse model of PcP-related IRIS 
. While both SSZ and anti-CD3 altered the T cell response to Pc and reduced immunopathogenesis, they produced differential outcomes with respect to fungal clearance. Anti-CD3 produced a profound inhibition of T cell responses which reduced disease, but also prevented the clearance of Pc from the lung. In contrast, SSZ dampened PcP-related immunopathogenesis without suppressing TH responses to a degree that prevented eradication of the organism. SSZ not only reduced T cell-mediated inflammation, but altered the nature of the T cell response by promoting TH2 lung cytokine environment and alternative activation of macrophages. It is likely that the preservation of TH2 responses combined with a shift in the polarization of AMs in SSZ-treated mice is responsible for the differential effects of SSZ and anti-CD3.
Another important aspect of our work is the development of a multispectral imaging flow cytometer-based method to assess the in vivo
phagocytic activity of AM during a T cell-mediated immune response by quantifying the percentage of AMs that contain internalized Pc. Understanding the mechanisms controlling Pc phagocytosis is an area of great interest, and many investigators have utilized various techniques to perform in vitro
assessments of Pc phagocytosis 
. However, demonstrating an in vivo
role for AM phagocytosis in the clearance of Pc has been more difficult. AM with associated Pc have been observed in the BAL fluid of patients and animals 
. However, this was in the setting of active PcP, the level of phagocytosis appeared low, and the significance to organism clearance was not determined. Others have performed in vivo
assessments of phagocytosis immediately (within 24 hours) following a bolus inoculation of labeled Pc 
. In addition, short-term depletion of AMs in rats reduced the clearance of Pc over the initial 24 hours post-inoculation 
. While these studies were able to demonstrate a role for AM in vivo
, the timing indicates that the investigators were evaluating the innate immune response to a bolus inoculation of Pc, rather than the CD4+
T cell-mediated response which is required for natural clearance of Pc from the lung. Using this new technology we were able to develop an assay to show that AMs are effector cells for the clearance of Pc during a natural CD4+
T cell-mediated immune response in vivo
. The advantages of these ImageStream-based data are that: 1) internalized Pc was distinguished from attached Pc; 2) a large number of AM from each animal was rapidly assessed to provide quantification of the phagocytic response; 3) the dependence of phagocytic activity on the presence of CD4+
T cells was demonstrated; and 4) the CD4+
T cell-dependent increase in phagocytic activity correlated with the clearance kinetics of Pc. Importantly, the ImageStream data was validated using confocal microscopy to co-localize intracellular Pc with the lysosome protein LAMP-1. These data indicate that Pc is located within the phagolysosome of AM, consistent with phagocytosis of the pathogen. The multispectral imaging flow cytometry technology should provide a valuable tool for further study of Pc phagocytosis in vivo
In summary, the results of this study indicate that the immune response to Pc can be modulated in a manner that reduces inflammatory consequences of PcP while enhancing the pathogen clearance through increased AM phagocytic capacity. We also developed a method for in vivo quantification of AM phagocytosis of Pc, and provide evidence that the macrophage is the ultimate effector for the CD4+ T cell-mediated clearance of Pc from the lungs. Immune modulation of T cell and AM functions should be considered potential therapeutic targets for the treatment of immune complications of PcP. Macrophages are equipped to recognize and eliminate pathogens as well as promote and/or resolve inflammation. Our results indicate that the phagocytic function of macrophages can be enhanced with a concomitant reduction in their pro-inflammatory properties. Enhancement of AM-mediated clearance of Pc may prove less inflammatory and generally superior to antibiotic therapy alone.