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1.
Figure 3

Figure 3. Myeloid KLF2 is Protective Against Endotoxemic Shock. From: The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock.

(A) Age and sex matched Lyz2cre and Lyz2creKlf2fl/fl mice were challenged with LPS (21mg/kg). These mice were observed for 72hr for survival.
(B–D) Age and sex matched Lyz2cre and Lyz2creKlf2fl/fl mice were challenged with LPS (21mg/kg) and monitored for changes in core body temperature, systolic blood pressure and shock index (SI=Heart Rate/Systolic Blood Pressure).
(E–J) Age and sex matched Lyz2cre and Lyz2creKlf2fl/fl mice were challenged with LPS (21mg/kg). Plasma was obtained four hours after LPS or saline administration by retro-orbital bleeding. Inflammatory cytokines for the indicated factors were analyzed using MILLIPLEX MAP mouse cytokine and chemokine panel from Millipore.

Ganapati H. Mahabeleshwar, et al. Immunity. ;34(5):715-728.
2.
Figure 2

Figure 2. KLF2 Negatively Regulates Bactericidal Activity of Macrophages. From: The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock.

(A and B) Peritoneal macrophages or neutrophils from Lyz2cre and Lyz2creKlf2fl/fl mice were inoculated with E. Coli at a MOI of 4 and intracellular bacterial killing analyzed by antibiotic protection assay as described in “Methods”.
(C and D) Primary peritoneal macrophages or neutrophils from Lyz2cre and Lyz2creKlf2fl/fl mice were stimulated with 100 ng/ml LPS for 12 hr. CRAMP and iNOS mRNA expression was analyzed by qPCR and normalized to 36B4.
(E and F) Peritoneal macrophages from Lyz2cre and Lyz2creKlf2fl/fl mice were induced with 100 ng/ml LPS for 0–12 hours. Cell lysates were analyzed for expression of iNOS by immunoblot. In a parallel experiment, cell culture supernatants following 20 hr of 100ng/ml of LPS treatment were analyzed for nitrite concentration by Griess assay as an index of iNOS activity.
(G and H) Lyz2cre and Lyz2creKlf2fl/fl mice peritoneal macrophages were stimulated with 100ng/ml LPS. Lactate concentration from these cell culture supernatants and intracellular ATP concentration from cell lysates were quantified and normalized to total protein content.
(I and J) Lyz2cre and Lyz2creKlf2fl/fl mice were stimulated with 100 ng/ml LPS. Total RNA was isolated and mRNA expression of Glut-1 and PGK was analyzed by qPCR.

Ganapati H. Mahabeleshwar, et al. Immunity. ;34(5):715-728.
3.
Figure 1

Figure 1. Myeloid KLF2 Deficiency is Protective Against Polymicrobial Infections. From: The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock.

(A). Lyz2cre and Lyz2creKlf2fl/fl mice plasma were obtain by retro-orbital bleeding and inflammatory cytokines were analyzed using MILLIPLEX MAP mouse cytokine-chemokine panel from Millipore.
(B) Age and sex matched Lyz2cre and Lyz2creKlf2fl/fl mice were subjected to CLP. These mice were observed for survival for 5 days.
(C and D) Age and sex Lyz2cre and Lyz2creKlf2fl/fl mice were subjected to CLP and their average systolic blood pressure and core body temperature measured following 24 hours of CLP.
(E–H) Blood, lung, liver and kidney were collected from Lyz2cre and Lyz2creKlf2fl/fl mice following 12 hours of sham or CLP procedure. Serial dilutions of these tissue homogenate were plated on tryptic soya agar plates and enumerations of bacterial colony forming units were indicated.
(I–L) Age and sex matched Lyz2cre and Lyz2creKlf2fl/fl mice were subjected to sham or CLP procedure. Plasma of these mice was obtained by retro-orbital bleeding and inflammatory cytokines were analyzed using MILLIPLEX MAP mouse cytokine and chemokine panel from Millipore.

Ganapati H. Mahabeleshwar, et al. Immunity. ;34(5):715-728.
4.
Figure 5

Figure 5. Impaired Cellular Motility and Metabolism in KLF2 Deficient Macrophages are HIF- 1α Dependent. From: The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock.

(A and B) Primary peritoneal macrophages from Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were stimulated with 100ng/ml LPS and added to the upper chamber of a migration or invasion tissue culture insert. Cells were allowed to migrate for 18hr and stained with Giemsa. The number of control, unstimulated cells migrated or invaded across the membrane was assigned as 100% and fold changes over this are indicated.
(C–L) Primary peritoneal macrophages or neutrophils from Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were stimulated with 100 ng/ml LPS. Total RNA was isolated and indicated target genes were analyzed by qPCR and normalized to 36B4. Expression of indicated genes in untreated control peritoneal macrophages was set as one.
(M and N) Peritoneal macrophages from Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were stimulated with 100ng/ml LPS. Lactate concentration from these cell culture supernatant and intracellular ATP concentration from cell lysates were quantified and normalized to total protein content.
(O and P) Peritoneal macrophages from Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were stimulated with 100 ng/ml LPS. Total RNA was isolated, Glut-1 and PGK mRNA expression was analyzed by qPCR and normalized to 36B4.

Ganapati H. Mahabeleshwar, et al. Immunity. ;34(5):715-728.
5.
Figure 4

Figure 4. KLF2 Inhibits LPS Induced NFκB Mediated HIF-1α Expression. From: The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock.

(A) Wild-type primary peritoneal macrophages were exposed to heat inactivated S. Aureus or E. Coli extracts in normoxic or hypoxic condition. Cell lysates were analyzed for KLF2 and HIF-1α protein expression by immunoblotting.
(B) Whole blood-derived RNA was obtained from control and patients with septic shock and subjected to microarray analysis. Relative expression of KLF2 and HIF-1α are indicated.
(C) Wild-type mice peritoneal macrophages were stimulated with increasing dose of LPS for 8h. Cell lysates were subjected to immunoblot using anti-HIF-1α antibody.
(D–F) RAW264.7 cells infected with Ad-GFP and Ad-KLF2 or primary peritoneal macrophages and neutrophils from Lyz2cre and Lyz2creKlf2fl/fl, mice were stimulated with 100 ng/ml LPS. Total RNA was isolated and HIF-1α mRNA expression was analyzed by qPCR.
(G) RAW264.7 cells transfected with the HIF-1α promoter luciferase construct were co-transfected with KLF2 or NFκB (p65) plasmid. These cells were stimulated with LPS and cell lysates were analyzed for luciferase activity.
(H and I) RAW264.7 cells infected with Ad-GFP or Ad-KLF2 or primary peritoneal macrophages from Lyz2cre and Lyz2creKlf2fl/fl mice were stimulated with LPS for 1hr. Chromatin immunoprecipitation was performed using the indicated antibody on the HIF-1α promoter containing the NFκB binding site at −197/−188 base pairs.
(J) Basal expression of HIF-1α in Lyz2cre and Lyz2creKlf2fl/fl mice peritoneal macrophages were analyzed by immunoblot.
(K and L) Primary peritoneal macrophages and neutrophils from Lyz2cre and Lyz2creKlf2fl/fl mice were stimulated with 100 ng/ml LPS. Cell lysates were analyzed for HIF-1α protein expression by immunoblot.

Ganapati H. Mahabeleshwar, et al. Immunity. ;34(5):715-728.
6.
Figure 6

Figure 6. Deficiency of HIF-1α Rescues Polymicrobial Infection and Endotoxic Shock Phenotypes in KLF2 Deficient Mice. From: The Myeloid Transcription Factor KLF2 Regulates the Host Response to Polymicrobial Infection and Endotoxic Shock.

(A) Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice plasma was obtained by retro-orbital bleeding and inflammatory cytokines analyzed using MILLIPLEX MAP mouse cytokine and chemokine panel from Millipore.
(B) Survival of age and sex matched Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice subjected to CLP. These mice were observed for 5 days for their survival following CLP.
(C and D) Blood and lungs were collected from Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice following 12 hours of sham or CLP procedure. Serial dilutions of blood and lung homogenate were plated on tryptic soya agar plates and enumerations of bacterial colony forming units were indicated.
(E) Peritoneal macrophages from Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were inoculated with E. Coli at a MOI of 2 and intracellular bacterial killing analyzed by antibiotic protection assay.
(F–I) Primary peritoneal macrophages and neutrophils from Lyz2cre, Lyz2creKlf2fl/fl Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were stimulated with 100 ng/ml LPS. Total RNA was isolated and both iNOS and CRAMP mRNA expression analyzed by qPCR and normalized to 36B4.
(J–L) Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were monitored for core body temperature, systolic blood pressure and survival after LPS challenge.
(M–P) Age and sex matched Lyz2cre, Lyz2creKlf2fl/fl, Lyz2creSetd2fl/fl and Lyz2creKlf2fl/flSetd2fl/fl mice were challenged with LPS (21mg/kg). Plasma of these mice were obtain four hours after LPS or saline administration by retro-orbital bleeding. Inflammatory cytokines for the indicated factors were analyzed using MILLIPLEX MAP mouse cytokine-chemokine panel from Millipore.

Ganapati H. Mahabeleshwar, et al. Immunity. ;34(5):715-728.

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