The normal host requires q an intact cell-mediated immune system - - PowerPoint PPT Presentation

The normal host requires q

• an intact cell-mediated immune system
• a sufficient number of, and normally functioning, neutrophils

to effect the normal response to the conidia we all continually encounter. The normal response is to clear conidia without establishment of an infectious nidus.

HOST DEFENSE vs. ASPERGILLUS

1. First line – pulmonary macrophages & monocyte-derived macs.

• a. Kill conidia, prevent germination by opsonin-independent

nonoxidative method and oxidative method

• b. Depressed by glucocorticoids (not through T)
• c. CGD can do
• d. Not inc. by IFNg
• d. Not inc. by IFNg
• e. Conidia resist oxidative

PMN also kill conidia by nonoxidative method (CGD PMN can). PMN also kill conidia by nonoxidative method (CGD PMN can). Lactoferrin seems important. In immunocompromised, PMN are critical at the time of challenge in the initial host response.

HOST DEFENSE vs. ASPERGILLUS

• 2. Second line – once germinated, PMN and monocytes kill hyphae
• a. Occurs even without opsonins
• b. Activation not needed, but could enhance
• c. Oxidative mechanism, defensins
• d. Depressed by corticoids (by dec. mobilization?); CSFs, IFNg

reverse this effect on PMN, monos

• e. CGD PMN can’t
• f. Monocytes principally use H2O2, CGD results conflict
• g. T cells can damage hyphae, & potentiate PMN

Serum is inhibitory at concs. >20% (Chiller et al.); controversial

N l Pl t l t New player- Platelets

S i (5HT) i id l idi & h h Serotonin (5HT) is cidal to conidia & hyphae Pl l dh & d f i Thi Platelets adhere to & spread over fungi. This activates the platelets (markers), & they degranulate This affects cell wall modeling, which damages h h h the hyphae.

• C. Lass-Florl
• C. Lass Florl

Additional points about host defenses and p Aspergillus Anti-defense by Aspergillus Anti defense by Aspergillus Conidia produce a catalase & an i hibi f id i b f inhibitor of mac. oxidative burst & of pro-inflammatory cytokine production H h d t h h li Hyphae produce proteases, phospholipases, gliotoxin (inhibits macs. & lyms.)

Innate mediators of host resistance: The Collectins Pattern recognition molec les The Collectins - Pattern recognition molecules These are collagenous carbohydrate-binding proteins Lung Surfactant Protein A (SP-A) and SP-D Mannose Binding Lectin-MBL

A ill i d C ll i SP A SP D Aspergillosis and Collectins- SP-A, SP-D

Bind to mannose & maltose Aggregate conidia

PMN oxidative burst

Function by binding to cell or conidia receptor? KO- SP-D more important Rx purified human SP-D or rSP-D

survival Steroid suppressed pul. murine model.

Aspergillus and collectins- MBL Aspergillus and collectins MBL

• MBL binds to conidia, agglutinates them, inc. C4b deposition,

i PMN h t i & id ti b t +/ i killi

• inc. PMN phagocytosis & oxidative burst, +/- inc. killing
• MBL deficiency (mice) didn’t affect survival, but mice have 2

types & may’ve had enough of other types, & may ve had enough of other Cortisone s ppressed p l aspergillosis

• Cortisone-suppressed pul. aspergillosis in BALB/c

– Rx with rhMBL i.n. enhanced survival and reduced pulmonary fungal burden

– Treated mice showed ↑TNFα, IL1β & IFNγ; ↓IL10 , β γ;

Suggests role for MBL in resistance and therapy

Kaur, et al. 2007. Clin Exp Immunol. 148:382-89.

Systemic Aspergillosis in MBL KO Mice

MBL KO and WT B6 mice Infected i.v. with A. fumigatus.

MBL KO mice were LESS susceptible to systemic MBL KO mice were LESS susceptible to systemic infection!

Further study needed to assess innate role of MBL in pulmonary disease

Clemons, et al. ICAAC 2008

Collectins

mannose binding lectin SP-A SP A SP-D pentraxin 3

may help phagocytes by acting as opsonins. Some also are directly cytotoxic cytotoxic. Production of chemokines may call on the appearance of other phagocytes at the local site. If, because of insufficiencies in phagocyte number or function a , p g y more durable response is needed, the innate immune system will become further engaged and initiate the engagement of the adaptive immune system immune system.

Triggering the innate immune system

Conidia as they germinate expose β-glucan β-glucan triggers the innate immune system thru D ti t i iti ti i fl t Dectin receptors, initiating an inflammatory response H ft i t h h h h t However, after conversion to hyphae, as hyphae mature the exposure of β-glucan decreases

The fungus is recognized by Toll-like receptors (TLR). This encounter in dendritic cells (DC) appears to be critical for This encounter in dendritic cells (DC) appears to be critical for subsequent coordination of the immune system. If idi h ’t b t i d b h t d If conidia haven’t been contained by phagocytes, and some germinated into hyphae, the latter may subvert the development of the appropriate response.

The attachment of the fungal forms to different DC receptors

• probably mannose receptors for conidia

& CR3 for hyphae & CR3 for hyphae

and the different methods of DC phagocytosis of the different fungal forms may lead to different DC signals and thus to different cytokine patterns thus to different cytokine patterns.

The DC take the internalized fungi to regional lymph nodes. The DCs mature express MHC II process antigen; which TLR path The DCs mature, express MHC II, process antigen; which TLR path triggered likely determines which co-stimulatory molecules are expressed, & thus whether Th1 or Th2 pathways ensue. The cytokines initially produced by the naïve phagocytes may also direct DC path. p

The adaptive immune system, largely represented by T cells, is engaged. p y ,

g y p y

, g g T cells, in context of MHC, become activated, and proceed to elaborate either Th1 or Th2 dominant cytokines thus determining the adaptive either Th1- or Th2-dominant cytokines, thus determining the adaptive response. Regulatory T cells likely have an important role in inhibiting the development of allergy.

Patients responding to R ha e strong Th1 Patients responding to Rx have strong Th1 lymphoproliferative responses and high IFNγ/IL-10 ratios ratios. Passive transfer

• f Th1 cells is protective

Passive transfer (animal expts.) of Th1 cells is protective.

The key cytokines associated with a successful y y response to Aspergillus infection are the proinflammatory and Th1 cytokines: p y y IFNγ

IL-1

TNFα

IL-6

IL-12 IL-18 GM-CSF

Their production, rather than down-regulation of Th2 cytokines IL-4 & IL-10, appears to be even more important. p .

Mononuclear cells produce IFNγ, GM-CSF, TNF & IL 2 i idi TNFα & IL-2 in response to conidia. Studies suggest Th-1 stimulating antigens are in gg g g conidia, hyphal components favor Th2 path (for hyphal components favor Th2 path (for both human & murine cells).

Th2

In contrast, progressive invasive infection is associated with enhanced production of IL-4, associated with enhanced production of IL 4, IL-10, IgE, and depressed IFNγ

IL-17 -new story!

Th17 cells now thought to be separate lineage of effector Th cells contributing to immune pathogenesis effector Th cells contributing to immune pathogenesis previously attributed to Th1 produce IL-17 promote neutrophil-mediated inflammation linked to resistance to several bacterial & parasitic infections also correlate with disease severity & immunopathology also correlate with disease severity & immunopathology

• L. Romani, Advances Against Aspergillosis 2008

Th17, not uncontrolled Th1, assoc. w. defective fungal clearance, failure to resolve inflammation, failure to i iti t t ti initiate protective responses. IL-17 & IL-23 inhibit PMN fungicidal activity & decrease inflammation even in presence of IFNg decrease inflammation even in presence of IFNg, suggesting Th17 prevails over Th1 Blocking IL-17 & IL-23 decreases PMN ingress, g g , prevents pathogenic inflammatory responses, ameliorates infection, restores protectiveTh1 antifungal resistance

One experimental way to study what genes are turned on in the immune response- example:

Transcription Analysis of Murine

turned on in the immune response example:

Transcription Analysis of Murine Kidney and Brain in Response to Systemic Aspergillosis

Juergen Loeffler1, Karl V. Clemons3, Michael Bonin2, g Sven Poths2, Hermann Einsele1, David A. Stevens3

1University of Wuerzburg, Medizinische Klinik II, Wuerzburg, Germany 2University of Tuebingen Microarray Facility Tuebingen Germany 2University of Tuebingen, Microarray Facility, Tuebingen, Germany 3Santa Clara Valley Medical Center, San Jose, CA, USA

Aims of the study:

• Perform genome-wide expression

profiling for transcriptional host-response in p g p p kidney and brain of mice infected with A. fumigatus g

• Compare gene expression profiles of

uninfected and infected mice uninfected and infected mice

• Compare expression profiles of infected mice to transcriptional response
• f human dendritic cells to A. fumigatus

Murine model - systemic aspergillosis Murine model systemic aspergillosis

Experimental Design Experimental Design Day 0 - Infect female CD-1 mice with 8 x 106 idi i f A f i t 10AF conidia i.v. of A. fumigatus 10AF Sample times - d 1 (ca. 14 h, PI), d 5, d 9 Tissues - brain and kidneys snap frozen Infected mice n 3 each time point Infected mice – n = 3 each time point Uninfected mice – n = 3

Microarray procedure

For analysis: Samples pooled ( 3/ l ti ) (n = 3/sample time) Mean values used to determine magnitude

• f change
• ligoDNA

g

• ligoDNA

Labeled cRNA

• ligoDNA

Day 1 PI - no inflammation or immune response genes showed differential regulation. Day 5 and 9 PI - inflammation or immune y response genes differentially regulated Differential expression of cytokine & chemokine genes in kidney and brain chemokine genes in kidney and brain Significantly fewer genes differentially regulated i b i th kid (b i d t ti in brain than kidney (brain needs protection against inflammation)

e.g., TNF pathway Genes in TNF-α pathway up-regulated (2- to 18-fold) in kidney only: ( ) y y

• TNFRSF1 (receptor)

S 9 ( )

• TNFRSF9 (receptor)
• TNFAIP3 (anti-apoptotic)
• TNF-α

TNF α

Pathway very important for effective innate immune response to A fumigatus (down immune response to A. fumigatus (down- stream of TLR2/4)

Neutropenia and Aspergillosis

Gene expression studies in immunocompetent mice infected with conidia indicate upregulation of mice infected with conidia indicate upregulation of Th1 genes and downregulation of regulatory T cells In neutropenic mice, both Th1 & Th2 genes are

• upregulated. This suggests that in neutropenia there
• upregulated. This suggests that in neutropenia there

is mis-regulation & an uncoordinated immune response

Corticosteroids in Aspergillosis

Long recognized as a risk factor for human invasive aspergillosis

Di t ff t t d h t Direct effect to depress phagocytes

Suppression of 1st line defense may relate to failure of phagolysosomal fusion Suppression of 2nd line defense mechanism?- conflicting data on oxidative burst Favorable effect of IFNγ & GM-CSF in reversing phagocyte-depressing effects

• f steroids on monocytes, polymorphonuclears & macrophages

Delay cytokine production Prevents monocyte & PMN recruitment to foci of infection Depresses TNF production D IL 12 RNA Depresses IL-12 mRNA

Effect of Antifungal Rx

AmB Rx aspergillosis associated with switch to Th1 profile with enhanced IFNγ and depressed IL-4 in serum with enhanced IFNγ and depressed IL 4 in serum Modification of TLR signaling with liposomal AmB. L i fl ti Less inflammation.

G-CSF synergizes with antifungal chemotherapy

Effect of Antifungal Rx

We evaluated the immunomodulatory effects of 3 different classes of antifungal h h i ll li THP 1 i l d i h idi i i agents on the human monocytic cell line, THP-1, stimulated with conidia in vitro. Amphotericin B, micafungin, and voriconazole reduced production of tumor necrosis factor (TNF) in conidia stimulated cells factor (TNF)- in conidia-stimulated cells.

The reduction of TNF- production correlated with TNF gene expression assessed by PCR and level of nuclear factor κB (NFκ B). Costimulation with GM-CSF abolished immunomodulatory effects.

Antifungal agents affect the immune reaction caused by A. fumigatus conidia in stimulated monocytes at clinically achievable concentrations. The impact of these immunomodulatory effects should be assessed.

J H Ch i E Y K C M P k S M Ch i D G L J H Y W S Shi D A S b i d J-H Choi, E-Y Kwon, C-M Park, S-M Choi, D-G Lee, J-H Yoo, W-S Shin, D A Stevens; submitted

Aspergillosis Th I it i E thi ? The Immunity is Everything?

Qualitative and quantitative Phagocyte defects

• Chronic lung disease

Innate immunity

Successful outcome of IA strongly related to

immune reconstitution Steinbach, 2005; Chandrasekar 2005

• Neutropenia
• Corticosteroids
• GVHD
• Chronic lung disease

Adaptive Anatomic barriers

Virulence

Transient clinical & radiologic worsening

with immune reconstitution [may be short]

Fungal burden

• GVHD

Environmental exposure

Adaptive immunity

with immune reconstitution [may be short]

Takuma, 2002; Caillot, 2001

Spontaneous resolution reported coinciding Spontaneous resolution reported coinciding

with immune reconstitution Protection Invasive Aspergi

A tif l R Th1

Immune Reconstitution

Antifungal Rx Th1 Reduce immunosuppression transplant Th1

But a RAPID shift to a proinflammatory reaction can Immune R tit ti S d (l k lik i di ) Reconstitution Syndrome (looks like worsening disease) Neutropenic pts. w. wbc to >4500 pul. complications & death

• G. Todeschini et al., Eur J Clin Invest 29:453

28% of pts reco ering from ne tropenia ha e a

• rsening

28% of pts. recovering from neutropenia have a worsening pulmonary picture and hypoxia. Occurs in 1st few days.

• M. Miceli, Cancer 110:112

Galactomannan titer helpful to distinguish from advancing infection infection

• N. Singh, Advances Against Aspergillosis 3

Augmentation of Host Response against Invasive Response against Invasive Aspergillosis

• Reverse immunosuppression where

possible

• Discontinue or rapidly taper
• Discontinue or rapidly taper

corticosteroids

• GM-CSF or G-CSF for persistent but

GM CSF or G CSF for persistent but reversible neutropenia

• Interferon-gamma
• Granulocyte transfusions for patients with

– refractory infection – persistent but reversible neutropenia

ImmunoRx of future?

• Infuse immune T cells?

Pulse dendritic cells Aspf16 cytotoxic Pulse dendritic cells Aspf16 cytotoxic lymphocytes cidal for Aspergillus

• G. Ramadan et al. Clin Exp Immunol 140:81

Th1 cells cultivated in vitro with Aspergillus antigen damage hyphae & potentiate antigen damage hyphae & potentiate PMN

• O. Beck et al. Blood 107:2562

Monoclonal antibody?

Mortality from Fungal Infections

GM-CSF Placebo 1/52 1.9% 9/47 19.1%

Rowe, et al. Eastern Cooperative Oncology Group Study, Blood

H GM CSF t id d i i How can GM-CSF reverse steroid depression in

• mac. antifungal function?

They act on cells via different receptors and i lli th signalling pathways. One possible common pathway: “the Choi h th i ” hypothesis”

Diagram 6 : Conidia + DEX + GM-CSF and NF-κB

Conidia I κB

Glucocorticoid

Toll-like receptor NF-κB

Receptor IKK

Degradation of IκB

Enhance NF-κB translocation Kinases

NF-κB

GM-CSF Cytokines

GM-CSF significantly antagonized DEX suppression of IL-1α and TNF-α production. (Ref) Kamberi M. et al., Cytokine 19:14, 2002

Improved definition of the Th1/Th2 balance is essential for future prospects for immunotherapy, antimicrobial chemotherapy and vaccination

THE END

Additional points about host defenses and p Aspergillus Pro-defense by Aspergillus Pro defense by Aspergillus Aspergillus antigens have adjuvant properties Stimulate CD4 & CD8 responses to another

• antigen. (& to self?)
• J. Tao et al. J. Leukocyte Biol. 80:529

Differentially regulated genes Day Kidney Brain Day postinfection Kidney

• No. genes

Brain

• No. genes

1 181 28 5 4524 383 5 4524 383 9 3758 1783 9 3758 1783 > 2–fold increase or decrease, P 2 fold increase or decrease, P <0.01

TNF & Aspergillosis

TNF deficient animals can’t control the early TNF-deficient animals can t control the early phases of infection. Neutralizing TNF with Ab reduces neutrophil influx into the lung & exacerbates infection into the lung & exacerbates infection. Administering TNF (e.g., intratracheally) enhances Administering TNF (e.g., intratracheally) enhances survival.

IFNγ & Aspergillosis

Neutralization of IFNγ exacerbates disease Neutralization of IFNγ exacerbates disease Giving IFNγ is protective Giving IFNγ is protective

IFNγ enhances neutrophil damage to hyphae, & reverses the

depressing effect of steroids on monocyte & neutrophil anti-hyphal activity. IFNγ upregulates neutrophil damage of hyphae even with CGD neutrophils. Giving IFNγ is associated with low IL-10 levels, & neutralization of IFNγ with elevated IL-10

Pulmonary aspergillosis & IFNγ y p g γ

• 1. We

found cortisone-suppressed DBA/2 i t t d ith ti IFN d i f t d i mice treated with anti-IFNγ and infected i.n. with A. fumigatus were not more susceptible than controls than controls.

• 2. IFNγ KO mice also not more susceptible to

systemic disease systemic disease.

• 3. Results differ from Cenci ‘99 – different

models? Mice? Strain of Aspergillus? models? Mice? Strain of Aspergillus?

IFNγ approved for use in CGD patients IFNγ approved for use in CGD patients.

IL-6 & Aspergillosis

IL-6 deficient mice are more susceptible A delay in IL-6 production leads to a decrease in A delay in IL-6 production leads to a decrease in neutrophil influx

IL-12 & Aspergillosis

Conidia appear to preferentially stimulate the IL-12 pathway of DC pathway of DC Neutralization of IL-12 exacerbates disease Neutralization of IL-12 exacerbates disease Therapy with IL-12 increases resistance Therapy with IL-12 increases resistance

IL-18 & Aspergillosis

Neutralization of IL-18 exacerbates disease

IL-18 deficiency results in down-regulation of TLR-2 and TLR-2 deficiency is associated TLR-2, and TLR-2 deficiency is associated with decreased TNF & IL-12 in response to Aspergillus infection Aspergillus infection

IL-4 & Aspergillosis

Neutralizing IL-4 enhances resistance Administering soluble IL-4 receptor, to antagonize IL-4, also results in resistance Administering IL-4 enhances susceptibility Mice deficient in IL-4 are resistant

they have increased IL-12 & decreased IL-10 & IgE; if the IL-12 is neutralized resistance is diminished emphasizing importance is neutralized, resistance is diminished, emphasizing importance

• f IL-12 over depression of IL-10

IL-4 depresses phagocyte damage to hyphae and the oxidative burst IL 4 depresses phagocyte damage to hyphae and the oxidative burst

Mouse Models of Aspergillosis

IL-10 in Fungal Infection Aspergillosis – in vivo models

• ABPA - regulation of cytokine response

ABPA regulation of cytokine response reduced mortality of sensitized WT mice, IL-10 KO mice show increased mortality IL-10 KO mice show increased mortality

• Systemic aspergillosis

IL 10 appears

• Systemic aspergillosis - IL-10 appears

deleterious to resistance, IL-10 KO mice show lower burdens & delayed mortality show lower burdens & delayed mortality

IL-10 & Aspergillosis

IL-10 levels are high in neutropenic pts. with aspergillosis Administering IL-10 enhances susceptibility Administering IL-10 enhances susceptibility Neutralizing IL-10 enhances resistance IL-10 KO mice are resistant

IL-10 deficient mice have increased inflammation at infection sites and increased levels of Th1 cytokines IL-10 may have a favorable regulatory role, dampening the harmful effects of inflammation

Mouse Models of Aspergillosis

CGD mice

gp91phox-/- (NADPH oxidase) gp91phox / (NADPH oxidase) Can establish pulmonary infection with A. fumigatus with no suppression; excessive fumigatus with no suppression; excessive inflammatory response even with sterile hyphae (Morgenstern, et al, ’97, J Exp Med) 47phox / p47phox-/- Pulmonary model nonsuppressed infected with A nidulans mutants (pH-insensitive) with A. nidulans mutants (pH insensitive) excessive inflammation in absence of fungi in tissues, also mortality by pABA mutant

(Bignell et al 05 IAI) (Bignell, et al, 05 IAI)

Mechanism of antagonism, by GM-CSF,

• f DEX suppression of mac killing of
• f DEX suppression of mac. killing of

conidia & proinflammatory cytokine production production.

• 1. Conidia dec. IKB cytoplasm & inc. NF-KB nuc. (both

by W. blot) y )

• 2. DEX inc. IKB cytoplasm & dec. NF-KB nuc., unstim. or

conidia-stim. mac. 3 GM CSF* dec IKB cytoplasm & inc NF KB nuc

• 3. GM-CSF* dec. IKB cytoplasm & inc. NF-KB nuc.,
• unstim. or conidia-stim. mac.
• 4. GM-CSF overcame DEX effects on IKB or NF-KB,
• unstim. or conidia-stim. mac.

*LPS pos. control

GM-CSF antagonism of DEX GM CSF antagonism of DEX suppression -corroborating evidence

Glucocorticoid acts on mac. anti-conidia activity thru the glucocorticoid receptor1 thru the glucocorticoid receptor The competition relates to killing, since glucocorticoids don’t affect phagocytosis2 glucocorticoids don t affect phagocytosis GM-CSF alone little effect on anticonidial activity3 β glucan in conidia may account for effect on β-glucan in conidia may account for effect on NF-KB, cytokine production4

• 1. JCI 76:1755; 2. RID 9(Suppl.3):S620, Res Immunol 149:454; 3. JLB 70:868, JID 187:705, Med Mycol 39:509, Cytokine 19:14, Microb Infect 4:133;
• 4. J Eukaryot Micro 48(Suppl):1603