A “Loss-of-Tolerance” Phenotype of the Liver Macrophage System to LPS Induces Overwhelming Systemic Inflammation and Early Death in a Knockout-Transgenic Mouse Model of Sickle Cell Disease.

Infections and death from fulminant sepsis remain a constant threat to patients with sickle cell disease (SCD). Established explanations include an impaired ability to contain and eliminate pathogenic organisms due to defects in spleen function and adaptive immunity. To the contrary, very little is known about the “first-line” response of the innate immune system after contact with pathogen associated cell components. An escalated immune (inflammatory) response to microbial structures would provide an alternative mechanism to explain increased rates of infectious complications and septic death in sickle cell patients. To test this hypothesis, knockout-transgenic mice homozygous for the human β-sickle globin gene (SS) were treated with a low dose of the canonical infectious stimulus lipopolysaccharide (0.5 μg/g bw; i.p.; 22°C) and compared to heterozygous sickle trait (ST) and C57BL/6 animals. Phenotypically, sickle mice appeared much sicker after LPS and displayed strict seclusion behavior, cessation of food intake, and physiological signs of stress. Body core (rectal) temperature decreased precipitously and irreversibly in sickle animals (~12°C/8hrs vs. ~2°C/8hrs [ST and C57BL/6]) followed by rapid death (50%/12h; 100%/48h vs 0% [ST; C57BL/6]). Analysis of the LD50 demonstrated an ~500-fold increased sensitivity to LPS in sickle mice (0.05 μg/g vs 25 μg/g [ST]). Serum cytokines (TNF-α, IL-6) were dramatically up-regulated in SS mice compared to control (TNF-α: 16-fold/2 hr post LPS, 100-fold/3 hr post LPS). Organ-specific immunohistochemical analysis of the marker cytokine TNF-α in liver, bone marrow, spleen, lung, and kidney four hrs after LPS revealed an astonishingly super-induced expression in the liver of sickle animals compared to controls. The liver of sickle animals showed several areas of coagulative liver necrosis unrelated to LPS and consistent with ischemic injury from recurrent sickle-mediated vascular occlusion. Immunoreactivity to TNF-α was most pronounced in areas of liver injury and mostly restricted to large macrophages (F4/80 +) surrounded by a T-lymphocytic (CD3+) infiltrate. In vitro analysis of Kupffer cells to serial concentrations of LPS recapitulated the in vivo results, demonstrating up to 20-fold larger TNF-α levels in cells derived from sickle livers. To further elucidate the role of the liver macrophage in the in vivo immune response to LPS, sickle animals were challenged with LPS forty-eight hrs after Kupffer cell depletion with Gadolinium Chloride. Sickle mice treated with Gadolinium experienced enhanced survival and an ~90% reduction in serum TNF-α levels. In summary, the present study offers new insights into the responsiveness of the innate immune system in SCD to the highly conserved bacterial cell component, lipopolysaccharide. Unexpectedly, these data suggest that the liver macrophage in SCD, typically a cell type tolerant to the pro-inflammatory effects of LPS, has a cardinal role in orchestrating an excessive and harmful innate immune response to bacterial infections. Further studies will have to determine the immune response to other conserved bacterial structures and relate these findings to the human form of SCD.