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IFI Neutrophiles vs Glukokortikoides
- 1. Neutropenia vs. glucocorticoids Insights into how immunosuppression may influence the pathology and pharmacotherapy of invasive pulmonary aspergillosis
- 2. Immune dysfunction Frequencyofaspergillosis Immune hyperactivity Frequencyofaspergillosis Acute IA Subacute IA ABPA Allergic sinusitis Slide: David Denning, University of Manchester Normal immune function Tracheobronchitis Aspergilloma Chronic cavitary Chronic fibrosing The Unique Interactions of Aspergillus with the Human Host
- 3. GVHD Neutropenia H&E 100x GMS 100x Representative Histopathology of IPA at Autopsy in Neutropenia vs. GVHD Chamilos et al. Haematologica 2006;91:986-9.
- 4. Glucocorticoid-induced immunosuppression Chemotherapy-induced neutropenia Cellular trafficking in BALF Rapid and extensive increase in PMN No influx of PMN TNF-α concentration in BAL Not detected High IL-10 concentration in BALF Low High Histological features Inflammation +++ No inflammatory exudate Necrosis with hyphae +++ Presence of fungal elements Small numbers of conidia Large numbers of invading hyphae Galactomannan levels in organs Low to very low High Efficacy of amphotericin B Similar to control (ineffective) Prolongs survival Dominant mechanism Adverse host response Fungal proliferation Differences in the Pathogenesis of Experimental Invasive Pulmonary Aspergillosis Balloy et al. Infection and Immunity 2005;73:494-503
- 5. Research questions How does the transcriptional profile of the host immune response differ between glucocorticoid versus neutropenic (chemotherapy) treated mice?
- 6. Intensive immunosuppression is required for IPA in Balb/c mice Pulmonary alveolar macrophages rapidly remove conidia in a neutropenic mouse without glucocorticoids GMS 400x H&E 1000x
- 7. Murine models of invasive pulmonary aspergillosis Day -4 -3 -2 -1 0 +1 +2 +3 +4 +5 Hydrocortisone 5mg X X X X X Cyclophosphamide 150 mg/kg plus X X hydrocortisone 5 mg (single dose) X Saline X X X X X Group #1 Group #2 Group #3 Intra-nasal inoculation under anesthesia A. fumigatus 293 (5x106 conidia) 1 hour (n=5) 24 hour (n=5) 72 hour (n=5) 120 hour (n=5) Female Balb/c 18-25 grams Animals euthanized and lung fixed (GMS, H&E staining) or flash-frozen in liquid nitrogen for PCR extraction
- 8. Gene expression profile Lungs were homogenized in lysis buffer and RNA isolated with commercial kits (RNAeasy, Qiagen) RNA quality confirmed by Agilent 2100 bioanalyzer Reverse transcription and real-time PCR was performed in triplicate at each timepoint in three mice for 84 immune response plus housekeeping genes RT2 Profiler PCR Array, Murine Innate and Adaptive Immune Response, Superarray Biosciences) Expression calculated by ∆∆ct method in relation to infected, non-immunosuppressed control animals
- 9. Neutropenic Glucocorticoid D+1 D+3 D+5 H&EGMSGMS H&E Histopathology Profiles in Experimental IPA High fungal burden, minimal inflammation Low fungal burden, extensive PMN influx
- 10. Relativeexpression(∆∆Ct)byRT-PCR vs.nonimmunosuppressedmice Immunopathology in glucocorticoid- immunosuppressed mice with IPA is associated with persistent suppression of “anti-inflammatory” cytokine expression cyclophosphamide glucocorticoid 72 hours (H&E) 72 hours (H&E) Neutropenic (cyclophosphamide) 0 24 48 72 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 IL-4 IL-10 IL-18 Time (hr) Steroid (hydrocortisone) 0 24 48 72 -7 -6 -5 -4 -3 -2 -1 0 1 2 3 4 5 IL-4 IL-10 IL-18 Time (hr)
- 11. Research questions How does the transcriptional profile of innate immune responses differ between glucocorticoid versus neutropenic (chemotherapy) treated mice? Can control of dysregulated inflammation improve outcome of experimental acute IPA in the glucocorticoid-immunosuppressed mouse?
- 12. Beyond drug carriers The potential for liposomes as immunomodulating agents Amphotericin B Liposome capsule Liposomal amphotericin B• Liposomes are phagocytosized by macrophages and PMN concentrate in the trans-golgi area – Similar site of localization for intracellular PAMP receptors (i.e TLRs 4, 7, 9) • Liposomal formulations of AMB have been shown to divert amphotericin B-d mediated signaling from TLR2 TLR-4, enhancing non-oxidative PMN effector functions critical for damaging Aspergillus hyphae Stimuli % Phagocyt. % Conidicid. O2 - (nM/106 ) TNF-α (pg/mL) IL-10 (pg/mL) None AMB-d L-AMB 30 + 3 39 + 4* 46 + 3* 22 + 3 39 + 4* 51 +7* 1.4 + 0.6 5.2 + 1.6* 1.7 + 0.5 220 + 18 361 + 22* 115 + 20* 39 + 3 41 + 4 155 + 10* *P< 0.05 vs. control Bellocchio et al. J Antimicrob Chemother 2005;55:214-222. Liposomes divert neutrophil response to Aspergillus conidia from pro-to anti-inflammatory
- 13. Murine models of invasive pulmonary aspergillosis Day -4 -3 -2 -1 0 +1 +2 +3 Hydrocortisone 5mg X X X X Cyclophosphamide 150 mg/kg plus X X hydrocortisone 5 mg (single dose) X Intra-nasal inoculation under anesthesia A. fumigatus 293 (5x106 conidia) Female Balb/c 18-25 grams Saline X X X X X X X X Empty liposomes X X X X X X X X Lipo AMB 10 mg/kg X X X X X X X X AMB-d 1 mg/kg X X X X X X X X Pre-treatment regimens
- 14. Comparison of lung injury and fungal clearance in corticosteroid-immunosuppressed mice with IPA 72 hour pretreatment with AmB-d, L-AMB, or empty liposomes AMB-d 1 mg/kg L-AMB 10 mg/kg Empty liposomes T=1h T=24h T=72h GMS 100x GMS 1000x 0 24 48 72 96 3 4 5 6 7 Control AMB-d L-AMB Empty Time (h) Log10AFCE(qPCR) A. fumigatus lung fungal burden Lewis et al. Antimicrob Agent Chemother 2007;52:1078-81. n=10 mice per treatment group
- 15. Comparison of survival in animals pre-treated with empty liposomes or AMB-d (n=10 mice per group) Lewis et al. Antimicrob Agent Chemother 2007;52:1078-81.
- 16. Ex vivo Aspergillus hyphal damage by Peripheral PMNs (1:1) (n=5 mice per group) Lewis et al. Antimicrob Agent Chemother 2007;52:1078-81. Baseline T=24h T=72h %hyphaldamage(XTT)
- 17. Ex vivo PMN Toll-like receptor expression versus glucocorticoid-immunosuppressed controls (n=5 mice per group, T= 72h) Lewis et al. Unpublished
- 18. Conclusions Profound differences in experimental acute IPA between the neutropenic and corticosteroid-immunosuppressed host PMN polarization and resolution of acute inflammation: Necrosis vs. apoptosis Modulation of dysregulated inflammatory /repair programs may improve outcomes of IPA in the glucocorticoid- immunosuppressed host Injury InflammationRepair
- 19. Acknowledgements Dimitrios P. Kontoyiannis Georgios Chamilos Nathan Albert Research support: Astellas Inc. Gilead Inc. Enzon Pharmaceuticals CLL foundation NIH-NCI Core 16672 EN Cobb Scholar Award
Editor's Notes
- Good Afternoon, On behalf of my colleagues, Drs Chamilos and Kontoyiannis, I would like to thank the committee for the opportunity to present our research. For nearly ten years, we have had an active collaborative research program focusing on translational questions surrounding the treatment of invasive fungal infections, particularly invasive aspergllosis—which remains a prominent causes of infectious diseases related morbidity and mortality at M.D. Anderson, particularly in the acute leukemia and hematopoetic stem cell transplant population
- Aspergillus is capable of causing a wide spectrum of diseases that can be functionally grouped on the status of the host immune response. At one extreme, patients who a persistently neutropenic of have suppressed cellular immunity due to transplantation or high-dose glucocorticoid therapy are prone to developing acute or sub-acute invasive forms of aspergillosis that generally start in the lungs but can disseminated to virtually any organ in the body if the patient remains persistently immunosuppressed. Crude mortality rates associated with IA range from 60-90%. At the other extreme, patients with immune hyperactivity to fungal spores are at prone to developing allergic bronchopulmonary aspergillosis or allergic sinusitis Patients with relatively normal immune function but pre-existing cavities or damage to the respiratory mucosa can be predisposed to saprophytic forms of the disease Unfortunately, little is known about how the pathology, diagnosis and treatment of invasive aspergillosis may change in patients with fluctuating immune function—a scenario that is often encountered frequently in patients at this institution
- In the largest contemporary autopsy study of invasive fungal infections in leukemia and HSCT recipients (n=314), our group found differences in the histopathological patterns of IPA in HSCT-recipients with GVHD versus persistently neutropenic patients. IPA in GVHD patient receiving high-dose corticosteroids was characterized by extensive lung inflammation and PMN infiltration, with less abundant hyphal burden. In contrast, neutropenic patients exhibited minimal inflammation but high fungal burden with extensive angioinvasion and tissue necrosis. Interestingly, both cohorts of patients had equally high prevalence of disseminated infection.
- These differences in the IPA pathology between the neutropenic and corticoidsteroid-immunosuppressed animals was further clarified by investigators at the Pasteur institute. Using mouse model of sinopulmonary aspergillosis, they compared the pathological features of the infection in neutropenic versus corticosteroid-immunosuppressed hosts In their model, the dominant mechanism of the adverse host response was a dysregulated inflammatory response in the lung compared to neutropenic animals, where unopposed fungal proliferation was the principal cause of damage to the host
- We were interested in further exploring these differences and in particular, trying to understand how these pathological differences between the glucocorticoid and neutropenic host may affect response to antifungal therapy We started be comparing transcriptional profiles of immune responses between neutropenic and glucocorticoid-immunosuppressed animals experimentally infected with Aspergillus fumigatus
- One of the challenges of this area is that many common mouse strains are highly-resistant (just like humans) to developing invasive aspergillosis. This is not surprising considering that mice constantly have their noses in ground! Balb/c mice have a tremendous capacity for sequestering and removing Aspergillus conidia or spores after an intranasal challenge. Even in neutropenic animals, inoculums in excess of 10 million conidia may be required to overwhelm pulmonary alveolar macrophages. This micrograph is from a neutropenic mouse challenged with 1x106 conidia neutropenic animals clearly shows that pulmonary alveolar macrophage defenses are intact and sequestering large amounts of conidia. To get around this problem, we had to administer a single dose of hydrocortisone even in neutropenic animals to achieve a reproducible infection and pathology, that simulates what was seen in the patient autopsy slides I showed earlier
- So the specifics of the experimental design are represented in this figure. 18-25 gram female balb C mice were immunesuppressed with either high dose hydrocortisone alone, or cyclophosphamide plus a single dose of hydrortisone to maintain neutropenia for 5 days. Mice were then inoculated intra-nasally under anaethesia with 5x106 Aspergillus fumigatus conidia. At serial timepoints mice were euthanized and lungs were immediately excised and flash-frozen in liquid nitrogen for either RNA isolation, of fixed in 10% neutral buffered formalin for and stained with H&E or GMS to compared patterns of diseased in the lung.
- RNA was isolated with commercial kits from Qiagen and the integrity was checked on an Agilent 2100 bioanalyzer before reverse transcription and amplification in the PCR arrays. Commercially available kits for profiling murine immune responses in the lung were used to evaluate gene expression profiles. Briefly, gene transcription and real-time PCR was performed in triplicate at each timepoint in three mice using commercial kitsthat detect 84 immune response genes plus housekeeping genes
- So looking at our results, we can see the clear histopathological differences in disease progression and host immune response between the neutropenic and glucocorticoid-immunosuppressed mice. Neutropenic mice developed areas of focal infiltrates containing extensive hyphal elements and minimal inflammatory exudate In contrast, patterns of infection in the glucocorticoid-immunosuppressed mice were characterized by diffuse inflammatory changes in the lung with extensive PMN influx, and minimal fungal proliferation. The clearest differences in the pathology were evident at 72 hours post-inoculation
- Comparison of the mean expression profiles in relation to control (non-immunosuppressed animals) reveals a divergent pattern of expression particularly at 72 hours when the most striking histopathological differences were evident. I am only showing data up to 72 hours because we are currently in the process of completing analysis of our last batch of lungs for Day 5 animals and this patterns appears to continue. Steroid-immunosuppressed mice exhibited a relative pattern of global suppression of chemokine, chemokine receptor, and both pro-and anti-inflammatory cytokine expression. This included key regulatory cytokines such as interleukin-4 and 10, which are important for dampening down innate inflammatory responses in the lung and initiating tissue repair. These studies would appear to confirm that high-dose glucocorticoid therapy disrupt key cytokine and chemokine regulatory networks that are required for an orchestrated and appropriate immune response to Aspergillus in the murine lung.
- This raised the question of whether this dysregulated inflammation could be controlled in the glucocorticoid-immunosuppressed host and improve the outcome of invasive pulmonary aspergillosis
- To explore this hypothesis, attempted to control this inflammation with unilamellar liposomes. Why liposomes? Because two decade of experience had shown that the marked pro-inflammatory activity of amphotericin B could be markedly reduced by incorporating the drug inside a liposome carrier. Liposomes have been shown to be one of the most potent agents at rescuing rats from endotoxemia after LPS challenge. Recent studies from Luigina Romani’s group in Italy also demonstrated that liposomal formulations of amphotericin B could actually enhance neutropenic phagocytic responses against Aspergillus, through non-oxidative mechanisms and boosting IL-10 production. While the imunomodulatory mechanism of liposomes is not entirely understood; several line of evidence suggest that liposomes through direct or indirect interactions with pattern recognition receptors divert Toll-like receptor activation from TLR-to TLR-4 responses, which stimulate non-oxidative effector functions in professional phagocytes
- Therefore we compared the relatively efficacy of empty (non-drug) containing liposomes, liposomal amphotericin B administered at 10 mg/kg, conventional amphotericin B deoxycholate, and control animals. We used a pre-treatement strategy because previous work with L-AMB demonstrated that liposome distribution to the lung was delayed by as much has 72 hours. Administration of the treatment regimens for 4 days prior to infection ensured lung tissue was loaded with the respective drugs prior to inoculation.
- So to get to the bottom line, AMB-D was ineffective at reducing fungal burden in glucocorticoid-immunosuppressed mice as we can see with this progressing diffuse consolidation in the lung with germinating hyphae. Animals treated with L-AMB 10 mg/kg or empty (non-drug) containing liposomes, however, developed only small focal infiltrates, inside these infiltrates only minimal germination of dimorphic small hyphal elements were visible. These histopathological results were confirmed by in our analysis of the Aspergillus lung fungal burden by quantitative real-time PCR, where the highest DNA tissue concentrations were observed in control and AMB-D treated animals. Remarkably, empty-drug free liposomes were nearly as effective as L-AMB dosed at 10 mg/kg day in clearing Aspergillus hyphae from the lung.
- Treatment continued until day +5 confirmed the efficacy of empty liposomes, which achieved 90% survival rates in glucocorticoid-immunosuppressed but not neutropenic animals. Only 50% of glucocorticoid-immunosuppressed mice treated with conventional amphotericin B survived until Day +5. So pre-treatment with an anti-inflammatory empty liposome was more effective in our glucocorticoid-immunosuppressed mouse model than administering a pro-inflammatory antifungal.
- In a second series of experiments we collected peripheral PMNs from glucocortioid-immunosuppressed mice infected with aspergillosis and pre-treated with saline, empty liposomes, or AMB-D at serial timepoints to assess neutrophil capacity to damage Aspergillus hyphae. Not surprisingly, PMNs from glucocorticoid-immunosuppressed mice exhibited a minimal capacity for hyphal damage that was augmented in AMD-D, empty liposome or Ambisome-treated mice. The superiority of the liposome formulations was consistent out to 72 hours
- Finally, similar to previous observations we saw patterns of increased TLR4, 7 and 9 expression in liposome pre-treated animals compared to AMB-deoxycholate treated animals.
- In conclusion, these studies suggest that at least in the setting acute experimental aspergillosis in mice, there are profound differences in the pathology of the infections between the glucocorticoid and neutropenic host. The plasticity of these responses may allow for novel immunomodulation strategies that could potentially improve outcomes and effectiveness of antifungal therapy in the non-neutropenic host. Currently we are exploring the potential of lipid mediators such as cyclopentenon prostoglandins to polarize neutrophils to a apoptotic pathways as an approach to control dysregulated inflammation and improve endothelial resistance to fungal invasion. These immunomodulation patterns may have additional utility for other acute inflammatory syndromes in the lung due to radiation or chemical, injury or other infections pathogens.
- I would like to acknowledge the contribution of my collaborators; Foremost Dimitrios Kontoyiannis George Chamilos who was instrumental in empty liposome studies and our technician, Nathan Albert who provided excellent technical assistance. Support for this study was provided by the following companies and funding mechanisms Thank you!