Sharon Erickson

Differential activation of the inflammasome by caspase-1 adaptors ASC and Ipaf.

Specific adaptors regulate the activation of initiator caspases; for example, FADD and Apaf-1 engage caspases 8 and 9, respectively. The adaptors ASC, Ipaf and RIP2 have each been proposed to regulate caspase-1 (also called interleukin (IL)-1 converting enzyme), which is activated within the ‘inflammasome’, a complex comprising several adaptors. Here we show the impact of ASC-, Ipaf- or RIP2-deficiency on inflammasome function. ASC was essential for extracellular ATP-driven activation of caspase-1 in toll-like receptor (TLR)-stimulated macrophages. Accordingly, ASC-deficient macrophages exhibited defective maturation of IL-1β and IL-18, and ASC-null mice were resistant to lipopolysaccharide-induced endotoxic shock. Furthermore, activation of caspase-1 in response to an intracellular pathogen (Salmonella typhimurium) was abrogated severely in ASC-null macrophages. Unexpectedly, Ipaf-deficient macrophages activated caspase-1 in response to TLR plus ATP stimulation but not S. typhimurium. Caspase-1 activation was not compromised by loss of RIP2. These data show that whereas ASC is key to caspase-1 activation within the inflammasome, Ipaf provides a special conduit to the inflammasome for signals triggered by intracellular pathogens. Notably, cell death triggered by stimuli that engage caspase-1 was ablated in macrophages lacking either ASC or Ipaf, suggesting a coupling between the inflammatory and cell death pathways.

Activation and accumulation of B cells in TACI-deficient mice

The tumor necrosis factor (TNF)-related ligand B lymphocyte stimulator (BLyS) binds two TNF receptor family members, transmembrane activator and calcium-modulating and cyclophilin ligand interactor (TACI) and B cell maturation molecule (BCMA). Mice that are transgenic for BLyS show B cell accumulation, activation and autoimmune lupus-like nephritis. The existence of at least two distinct BLyS receptors raises the question of the relative contribution of each to B cell functions. We therefore generated mice that were deficient in TACI. TACI−/− mice showed increased B cell accumulation and marked splenomegaly. Isolated TACI−/− B cells hyperproliferated and produced increased amounts of immunoglobulins in vitro. In vivo antigen challenge resulted in enhanced antigen-specific antibody production. Thus, TACI may play an unexpected inhibitory role in B cell activation that helps maintain immunological homeostasis.

APRIL-deficient mice have normal immune system development

ABSTRACT APRIL (a proliferation-inducing ligand) is a member of the tumor necrosis factor (TNF) superfamily. APRIL mRNA shows high levels of expression in tumors of different origin and a low level of expression in normal cells. APRIL shares two TNF receptor family members, TACI and BCMA, with another TNF homolog, BLyS/BAFF. BLyS is involved in regulation of B-cell activation and survival and also binds to a third receptor, BR3/BAFF-R, which is not shared with APRIL. Recombinant APRIL and BLyS induce accumulation of B cells in mice, while BLyS deficiency results in severe B-cell dysfunction. To investigate the physiological role of APRIL, we generated mice that are deficient in its encoding gene. APRIL−/− mice were viable and fertile and lacked any gross abnormality. Detailed histological analysis did not reveal any defects in major tissues and organs, including the primary and secondary immune organs. T- and B-cell development and in vitro function were normal as well, as were T-cell-dependent and -independent in vivo humoral responses to antigenic challenge. These data indicate that APRIL is dispensable in the mouse for proper development. Thus, BLyS may be capable of fulfilling APRILs main functions.

Heregulin, but not ErbB2 or ErbB3, heterozygous mutant mice exhibit hyperactivity in multiple behavioral tasks

Genetic redundancy is a problem in gene targeting studies because functionally relevant sister proteins can compensate for the lack of protein product of a targeted gene. A molecular system is chosen in which it is hoped to demonstrate both the lack and presence of compensation after disruption of particular single genes. Mammals may not be able to compensate for the lack of heregulin, a single ligand for multiple ErbB receptors, however, compensation is expected when a single ErbB receptor is knocked out. To investigate this the heregulin-1, ErbB2, or ErbB3 locus was disrupted in a targeted manner and mice heterozygous for the mutation were analyzed. Heregulin and its receptors were shown to be involved in embryonic brain development and, more recently, in plastic changes associated with adult brain function in rodents. Although they have never been shown to play roles in mammalian behavior, it was decided to characterize the mice behaviorally using a battery of simple tests. Heregulin mutant mice exhibited elevated activity levels in the open field, showed improved rotorod performance, and finished T-maze spontaneous alternation task faster compared to control wild type littermates, findings that suggest a consistent hyperactivity across tests. ErbB2 and ErbB3 mutant mice, whose strain origin was identical to that of heregulin mutants, showed no sign of the behavioral alterations. It is suggested that the abnormalities seen in heregulin mutant mice are due to mutation at that locus and the lack of alterations seen in ErbB2 and ErbB3 mutant mice is the result of compensation by unaltered sister receptors.

HER2-Targeted Therapy Reduces Incidence and Progression of Midlife Mammary Tumors in Female Murine Mammary Tumor Virus huHER2-Transgenic Mice

Purpose: This study examined the effectiveness of early and prolonged mu4D5 (the murine form of trastuzumab/Herceptin) treatment in transgenic mice that overexpress human HER2 (huHER2), under the murine mammary tumor virus promoter, as a model of huHER2-overexpressing breast cancer. Experimental Design: Mice were randomly assigned to one of three treatment groups and received i.p. injections from 17 weeks of age until either 52 weeks of age or morbidity. Fourteen mice received 100 mg/kg mu4D5, 14 mice received 100 mg/kg antiherpes simplex virus glycoprotein D control antibody, and 11 mice received a diluent control. Results: High levels of huHER2 expression were detectable in mammary glands of young virgin founder mice. Mammary adenocarcinomas were frequently found in female founders and progeny at an average age of 28 weeks, with some progressing to metastatic disease. The incidence of mammary tumors was significantly reduced, and tumor growth inhibition was observed in mice receiving mu4D5 compared with control mice. In addition, Harderian gland neoplasms, highly associated with overexpression of huHER2 in this transgenic line, were entirely absent in the mu4D5 treatment group, indicating down-regulation of huHER2 in vivo activity. Conclusions: Early intervention with mu4D5 was of benefit in our transgenic mice at high risk for developing huHER2-overexpressing breast cancer. This study suggests a potential benefit of early treatment with Herceptin in HER2-positive primary breast cancer.

Effects of in Vivo Heregulin β1 Treatment in Wild-Type and ErbB Gene-Targeted Mice Depend on Receptor Levels and Pregnancy

Mice heterozygous (+/-) for either heregulin (HRG), ErbB2, or ErbB3 were created by gene targeting, resulting in the loss of one functional gene copy and an associated decrease in targeted protein. We examined the in vivo activity of recombinant HRG peptide, rHRG beta1 (amino acids 177 to 241), in the three heterozygous mouse lines and in wild-type (WT) mice, both pregnant and nonpregnant. Nonpregnant WT and HRG(+/-) mice of both sexes were sensitive to rHRG beta1 treatment as evidenced by a high mortality rate associated with abdominal enlargement and parietal cell loss. However, pregnant WT mice and ErbB2 and ErbB3 heterozygous mice treated with rHRG beta1 were less affected, with significantly lower mortality rates and a less severe abdominal phenotype. Histological analysis revealed extensive breast ductal hyperplasia in females of all genotypes after rHRG beta1 treatment. Hyperplasia of other epithelial tissues such as the pancreas and intestine and the growth of cardiac nerve bundles were also observed, independent of sex.