Endogenous modulators of inflammation and its impact on infectious diseasesUlrich Matt1,2, Sylvia Knapp1,2

1Research Center for Molecular Medicine of the Austrian Academy of Sciences (CeMM), Vienna, Austria; 2Department of Medicine I, Div. of Infectious Diseases and Tropical Medicine, Medical University Vienna, Vienna, Austria;

Fig. 250 µl endotoxin-free HCl (red) or NaCl (=controls, blue) was instilled intratracheally in mice followed byintranasal infected with CFU P. aeruginosa i.n., and survival was monitored (A) or 16hrs after infection micewere sacrificed and lung CFUs were determined on blood agar plates (B). Data are mean ± SEM; *p <0.05, **p < 0.01

Fig. 4Endotoxin-free HCl was instilled intratracheally in mice followed by i.p. injection of Bß15-42-NH2 or sterile NaClat t=0, t=+1h and t=+6hrs. Control groups received sterile NaCl intratracheally and i.p. (A) After 24h alveolarmacrophages were harvested, and uptake of P. aeruginosa was assessed by FACS; (n=7 mice/group). (B)Representative slides from immuno-histochemical staining of IRAK-M on alveolar macrophages 24hrs after acidaspiration. (C) Lungs were harvested after 24hrs to conduct RT-PCR on IRAK-M; (n=3 mice/group). Valuesare expressed as mean ± SEM; *p < 0.05, ***p < 0.001.

Conclusions:1) The fibrin-derived peptide Bβ15-42 exerted protective effects during acid

aspiration, resulting in reduced vascular leak, and diminished inflammation.2) Acid aspiration aggravates subsequent Pseud. aeruginosa pneumonia.

Treatment with the fibrin-derived peptide Bβ15-42 during acid aspirationattenuates lung injury, and thereby improves outcome in subsequentPseud. aeruginosa pneumonia.

3) Later resolution of inflammation as suggested by elevated pro-inflammatorycytokine levels and concomitantly higher IRAK-M levels in untreated mice,which underwent acid apsiration impairs phagocytosis, and thus contributesto impaired survival in secondary bacterial infection.

Acid-induced inflammation impairs bacterial phagocytosis by alveolar macrophages

AKAP-inhibition prevents detrimental effects of OxPAPC in vitro and in vivo

Acid aspiration impairs host response to P. aeruginosa pneumonia

OxPAPC activates PKA distinctly

Fig. 6(A) RAW 264.7 cells were treated with increasing dosis of OxPAPC compared to DMPC, (B) incubated with DMPC(=control), or OxPAPC, with or without pre-incubation of Cytochalasin D (preventing actin polymerization). Cells weresubsequently stained for F-actin (phalloidin; green) and PI (red); Scrambled contol (Control) and shRNA transfected cellswere incubated with DMPC or OxPAPC and stained for actin cytoskeleton (C), or assessed fpr phagocytosis of FITC-labeledE. coli by FACS analysis (D). Uptake of bacteria is expressed relative to carrier. Data are mean ± SEM; * indicates p <0.05 versus corresponding carrier.

Fig. 1Endotoxin-free o,1N HCl (50µl) was instilled intratracheally in mice followed by i.p. injection of Bß15-42-NH2 or carrier (NaCl) at t=0 and t=+1h. Time course analysis for PMN influx (A), IL1β (B),and Itotal protein content (C) in BALF are depicted. n=6-8 mice/group; data expressed as mean ±SEM; *p < 0.05,

Fig. 9(A) Survival of PTENMC-KO and PTENMC-Wt mice infected with S. pneumoniae (n=11-13 per group). (B)Phagocytosis of S. pneumoniae by alveolar macrophages from PTENMC-KO and PTENMC-Wt mice; nuclei are inblue, ingested bacteria (green) are defined by co-localization with lysosomes (red) and appear yellow. (C)and (D) PTENMC-KO and PTENMC-Wt mice infected with S. pneumoniae. (C) lung inflammation 48h afterinfection, (D) TNF levels in BALF 6h post-infection. Data are presented as mean ± SEM. Statisticalsignificance (p value <0.05) is indicated by *.

Bbeta(15-42) protects against acid-induced

acute lung injury and secondary pseudomonas pneumonia in vivo

Bß15-42-NH2 dampens acid induced lung inflammation

Fig. 350µl endotoxin-free HCl, or NaCl (=controls, blue) was instilled intratracheally in mice followed by i.p. injection ofBß15-42-NH2 (green) or sterile NaCl (red) at t=0, t=+1h and t=+6h. After 24hrs all three groups were infectedwith P. aeruginosa i.n. Sixteen hours after infection mice were sacrificed and lung CFUs (A) and cytokines in lunghomogenates (B) were evaluated. n=6-9 mice/group. Data are mean ± SEM; *p < 0.05, **p < 0.01

WAVE-1 anchors PKA to facilitate the detrimental effects of oxidized phospholipids during Gram negative sepsis

Oxidized Phospholipids (OxPAPC) increases mortality and facilitates bacterial outgrowth and dissemination in E. coli peritonitis

Fig. 5Mice received i.p. either OxPAPC (green) or unoxidized control lipids (DMPC; black) and were i.p. infected with E. coli. (A)Survival data are representative of two independent experiments of n = 12/group; p value indicates the difference betweensurvivals by log rank test. (B) In separate experiments, mice were treated as above and bacterial outgrowth in peritoneal lavagefluid was determined 20hrs after infection. Data are mean ± SEM of n = 8 mice/group. Results are representative of twoindependent experiments. *, p < 0.05 vs control mice.

F-actin: phalloidin-FITC

Nuclei: propidium iodide

Bß15-42-NH2 treatment of aspiration-induced ALI dampens inflammation during secondary P. aeruginosa pneumonia

PK

A

+ -

co

ntr

ol

OxP

AP

CD

MP

CF

ors

ko

lin

PKA-kinase assay:

OxPAPC inhibits phagocytosis via a PKA-dependent actin-spread

Control 6-Bnz-cAMP forskolin OxPAPC

Phagocytosis assay:

loading control

Phagocytosis assay:

Fig. 7(A) In RAW 264.7 cells PKA kinase activity was âssessed after incubation with carrier, OxPAPC, DMPC or forskolin. RAWcells were incubated with DMPC (=control), OxPAPC, forskolin, or 6-Bnz-cAMP (=specific PKA-activator) and phagocytosis-assay (B) or staining for actin cytoskeleton (C) was performed. Data are mean ± SEM; * indicates p < 0.05, versuscorresponding carrier. (D) A-kinase anchoring proteins tether PKA to specific sites within the cell to fine tune its activity (fromScott and Pawson, 2000).

Fig. 7(A) RAW 264.7 cells were incubated with DMPC or OxPAPC alone or after treatment with 100µM Ht-31 (AKAP-to PKAbinding inhibitor) and stained as described. (B) RAW 264.7 cells were treated with carrier or phospholipids alone, or afterpreincubation with Ht-31 and phagocytosis was performed as described. Uptake is expressed relative to carrier and * indicatesp < 0.05. Data are representative mean ± SEM. (C) Mice received carrier or OxPAPC i.p., and/or Ht-31, immediatelybefore infection with E. coli. At t=10h PLF was harvested and bacterial CFUs enumerated. Data are mean ± SEM from n=7-9mice/group; * indicates p < 0.05 versus carrier. Or: (D) Survival was monitored every; n=12 mice/group, p value of eachexperiment is indicated.

Myeloid PTEN promotes inflammation but impairs

bactericidal activities during murine pneumococcal pneumonia

WAVE-1 mediates antiphagocytic properties of OxPAPC in vitro and in vivo

0 24 48 72 96 120 144 1680

25

50

75

100

PTENMC-KO

PTENMC-Wt

p=0.02

Time (h) after infection

Su

rviv

al (%

)

WT KO

Lung-inflammation

Phagocytosis

Cytokine production

S.pneumoniae-FITC

Lysotracker - Red

Dapi

Fig. 8(A) AKAPs known to associate with the actin-cytoskeleton: AKAP-Lbc (150bp), WAVE-1 (116bp) and Gravin(136bp) mRNA expression in primary peritoneal macrophages; GAPDH (372bp). (B) Primary peritoneal macrophagesof WT and WAVE-1-/- mice incubated with DMPC or OxPAPC and stained with phalloidin (green) and PI (red).(C) Phagocytosis of E. coli by WT and WAVE-1-/- peritoneal macrophages analyzed after prior incubation withDMPC or OxPAPC. Data are mean ± SEM; * p < 0.01 versus corresponding DMPC. (D) Innate differences in sizeof WAVE1-KO mice and wild-type animals. Bone marrow transplanted mice (E) und (F): WT and chimeric WAVE-1-/- mice were treated with DMPC or OxPAPC i.p. and infected E. coli i.p. (E) Peritoneal CFU counts wereenumerated 10hrs after infection, and (F) survival was monitored. Data are from n=9-12 mice/group and presented asmean ± SEM; * p < 0.05 versus corresponding DMPC control.

Conclusions:1) Oxidized Phospholipids (OxPAPC) inhibit phagocytosis and thereby impair

survival in E. coli peritonitis.2) OxPAPC activates PKA leading to an actin-spread, which inhibits

phagocytosis.3) Random PKA activation cannot inhibit phagocytosis or induce an actin

spread, instead OxPAPC activates PKA via the AKAP WAVE-1.

Myeloid PTEN augments inflammation and impairs bacterial killing during S. pneumonia pneumonia

PTENMC-Wt

PTENMC-KO

0

500

1000

1500

2000

*

KC

(p

g/m

l) lu

ng

PTEN MC-Wt

PTEN MC-KO

0

1

2

3

4*

ne

utr

op

hil

s x

10

6/m

l

PTENMC-WT

PTENMC-KO

0

10

20

*

ne

utr

op

hil

s x

10E

3/m

l B

AL

F

lung peritoneum

PTEN MC-Wt

PTEN MC-KO

2

3

4

5

6

log

CF

U/m

l B

AL

F

Despite reduced PMN recruitment in myeloid PTEN deficient mice, bacterial outgrowth is kept in check in theses animals during S. pneumonia pneumonia

Fig. 10PTENMC-KO and PTENMC-Wt mice were infected intranasally (A) or intraperitoneally (B) with S. pneumoniae andafter 6hrs PMNs were enumerated in lavage fluids. (C) KC levels in lungs were determined by ELISA. (D)65hrs after infecton, bacterial loads in lung homogenate was determined. Data are presented as mean ± SEM.Statistical significance (p value <0.05) is indicated by *.

Conclusions:1) Myeloid PTEN increases the inflammatory response, and simultaneously

impairs bacterial killing during pneumococcal pneumonia.2) While PTEN limits recruitment of PMNs to the peritoneum upon

pneumococcal infection, it increases their numbers in the alveolar spaceduring pneumonia.

3) The net outcome of myeloid PTEN deficiency is beneficial inpneumococcal pneumonia, as increased bactericidal activity and dampenendinflammation can compensate for the lack of PMNs.

A B C D

A B

C D

A B C

D E F

A B

C D E

A B

C D

A BA B C

A B

A B

A B

CD

A

C

B