Platelet factor 4 regulation of monocyte KLF4 in experimental cerebral malaria

Abstract

Cerebral malaria continues to be a difficult to treat complication of Plasmodium falciparum infection in children. We have shown that platelets can have major deleterious immune functions in experimental cerebral malaria (ECM). One of the platelet derived mediators we have identified as particularly important is platelet factor 4/CXCL4. Our prior work demonstrated that PF4(-/-) mice are protected from ECM, have reduced plasma cytokines, and have reduced T-cell trafficking to the brain. We now show that PF4 drives monocyte cytokine production in a Kruppel like factor 4 (KLF4) dependent manner. Monocyte depleted Plasmodium berghei infected mice have improved survival, and KLF4 is greatly increased in control, but not monocyte depleted mice. PF4(-/-) mice have less cerebral monocyte trafficking and no change in KLF4 expression. These data indicate that PF4 induction of monocyte KLF4 expression may be an important step in the pathogenesis of ECM.

Figure 1. PF4 Stimulates Monocyte Activation.

A. Dose Response. Monocytes were incubated with PF4 for 48 hrs and TNFα measured by ELISA (n = 4 ± S.D. *P<0.03 vs 0). B. Monocytes were treated with control PBS, 1 µg/mL PF4, or 50 U/mL heparin prior to PF4 as control. TNF-α was measured 48 hrs later (n = 4 ± S.D. *P<0.01 vs Control). C. IL-6. IL-6 was measured 48 hrs after PF4 by ELISA (n = 4 ± S.D. *P<0.01 vs control). D. Platelet PF4 increases monocyte IL-6 production Mouse monocytes were incubated with resting WT platelet supernatant and activated WT or PF4^−/− platelet releasate. 24 hrs later IL-6 was measured (n = 4 ± S.D. *P<0.01). E. Human monocyte cell line. THP-1 cells were incubated with 1 µg/mL of PF4 or PF4 in the presence of heparin and TNFα production measured (n = 3 ± S.D. *P<0.01 vs Control).

Figure 2. PF4 Increases Monocyte KLF4 Expression.

A. Mouse monocytes were incubated with 1 µg/mL of PF4 and KLF4 mRNA was quantified by qRT-PCR (n = 4; ± S.D. *P<0.05 vs Control). B. KLF4 immunoblot (n = 4 pooled samples). C. KLF4 Time Course. THP-1 cells were incubated with 1 µg/mL of PF4 and at each time point KLF4 expression was determined by immunoblot (representative image). D. Western blot quantification of KLF4 (n = 3; ± S.E.M *P<0.05 vs Time 0).

Figure 3. PF4 Stimulates KLF4 Activity.

A. PF4 induces KLF4 DNA binding. THP-1 cells were incubated with 1 µg/mL of PF4 and at each time point nuclear extracts isolated for ChIP. DNA was prepared and PCR amplified with primers for the known positive control bradykinin 2 receptor promoter. Control DNA and control total spleen cDNA were used as positive controls (far right). B. IL-6 Promoter Binding. KLF4 ChIP DNA was prepared and PCR amplified with primers for the IL-6 promoter. C. KLF4 mediates PF4 induced monocyte stimulation. THP-1 cells were treated with control siRNA or KLF4 siRNA and incubated or not with 1 µg/mL PF4. IL-6 was measured by ELISA (n = 4 ± S.D. *P<0.01 vs control).

Figure 4. Monocytes are Important in the Development of ECM.

A. Survival Curve. Mice were monocyte depleted with anti-CD14 antibody and infected with P. berghei (n = 5 *P<0.05 vs IgG). B. Monocytes are a significant source of KLF4 in ECM. mRNA. qRT-PCR for KLF4 was performed on spleens of uninfected and infected IgG and anti-CD14 treated mice (n = 5, S.D. *P<0.01 vs IgG) C. Protein. Spleen lysates were pooled and immunoprecipitated with anti-KLF4 antibody prior to KLF4 immunoblot (n = 5 mice).

Figure 5. PF4 Mediates KLF4 Expression in ECM.

A. qRT-PCR for KLF4 was performed on spleens from infected WT and PF4^−/− mice and expressed as fold change vs control uninfected mice (n = 5, S.D. *P<0.01 vs IgG). B. PF4 drives monocyte trafficking to the brain in ECM. Infected WT and PF4^−/− mouse brains were isolated on day 5 post infection and mononuclear cells isolated and total monocyte numbers determined (n = 5, ± S.D. *P<0.05). C. PF4 drives monocyte trafficking to the brain in ECM. Infected WT and PF4^−/− mice were given 1×10⁶ GFP positive monocytes on day 4, and 12 hrs later brains harvested and mononuclear cells isolated. GFP positive cells were quantified by FACS (n = 5, ± S.D. *P<0.03).