Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) induce cystitis, pyelonephritis, and can cause kidney scarring and failure if inflammation is not under control. The detailed effects of cytotoxic necrotizing factor 1 (CNF1), the key UPEC toxin, on the pathogenicity of UPEC remain unclear. CD36 is an important scavenger receptor, responsible for pathogen and apoptotic cell clearance, and plays an essential role in host immune defense and homeostasis. Regulation of CD36 by bacterial toxins has not been reported. In this study, using a pyelonephritis mouse model, CNF1 was observed to contribute to increasing neutrophils and bacterial titers in infected bladder and kidney tissues, resulting in severe inflammation and tissue damage. CD36 expression in macrophages was found to be decreased by CNF1 in vitro and in vivo. We demonstrated that CNF1 attenuated CD36 transcription by decreasing expressions of its upstream transcription factors LXRβ and C/EBPα and their recruitment to the CD36 promotor. In addition, Cdc42 was found to be involved in CNF1-mediated downregulation of LXRβ. Our study investigated the pathogenesis of cnf1-carrying UPEC, which affected host innate immune defenses and homeostasis through regulation of CD36 in macrophages during acute UTIs.

CNF1 is a key toxin secreted by UPEC, which induces inflammation during UPEC infections. CNF1 is well known to activate Rho GTPases to disturb host cell signaling pathways.

Inflammatory bowel disease (IBD) affects millions of Americans, and costs 1.7 billion annually. High mobility group box 1 (HMGB1) is a cytokine mediator of inflammation in the pathogenesis of IBD. Elevated levels of HMGB1 have been observed in human IBD and in mouse models of colitis (Vitali R, et al. Am J Gastroenterol. 2011. Yamasaki H, et al. Mol Med Report. 2009). Blocking HMGB1 induction by either anti-HMGB1 antibody or ethyl pyruvate (HMGB1 inhibitor) was able to ameliorate the inflammation in colitis mice. DNA with certain sequences or structures has been shown with high affinity binding to HMGB1, and has been reported to attenuate inflammatory responses by blocking HMGB1. In this study, we conjugated DNA sequences to sepharose beads and utilized them as novel reagents to remove HMGB1. We found that the DNA-beads were able to bind HMGB1 with a capacity of 7.6 μmol/L, and each immobilized DNA molecule was able to bind three HMGB1 molecules (DNA:HMGB1 ratio = 1:3). DNA-beads were able to capture HMGB1 from RAW 264.7 cell supernatant stimulated with LPS and from sera of septic mice induced by cecal ligation and puncture. When applied to stools of mice with dextran sulfate sodium (DSS) induced colitis, DNA-beads were able to capture HMGB1 from stools of these colitis mice. Hence, our data suggest a therapeutic potential for DNA-beads to remove HMGB1 in inflammatory diseases such as IBD.

Severe sepsis accounts for over 200,000 deaths in US annually (Angus et al, CCM, 2001). The mechanism involves endogenous molecules that impair organ function. Extracellular hemoglobin is one such factor enhancing tissue damage during sepsis. We designed haptoglobin-coated beads for a perfusion system to remove hemoglobin in an experimental sepsis model. Surprisingly, we observed that in addition to removing hemoglobin, haptoglobin beads also captured large amounts of HMGB1. HMGB1 is a major mediator on the final common pathway to lethality in sepsis (Yang et al, BBA 2009). Addition of haptoglobin suppressed HMGB1-stimulated TNF and IL-8 release in cultured macrophages. Haptoglobin knockout mice subjected to cecal perforation-induced sepsis had significantly higher serum HMGB1 levels and higher mortality compared to wild type animals (75% survival in wild type vs. 34% in haptoglobin knockout mice; n=15/group, P<0.05). Treatment with anti-HMGB1 antibodies significantly reduced the mortality in haptoglobin knockout mice (12% survival in control vs. 56% in animals receiving HMGB1 antibodies; n=16/group, P<0.05). Structure-function analysis revealed that haptoglobin beta subunit alone is sufficient to recapitulate the protective effects observed with haptoglobin. These findings indicate that haptoglobin is an endogenous modulator of HMGB1 that is capable of reducing the toxicity of HMGB1 in sepsis. Supported in part by grants from NIH (RO1GM62508, to KJT and RO1GM098446, to HY).

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