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The impact of Cryptosporidium parvum infection on host cell gene expression was investigated by microarray analysis with an in vitro model using human ileocecal HCT-8 adenocarcinoma cells. We found changes in 333 (2.6%) transcripts at at least two of the five (6, 12, 24, 48, and 72 h) postinfection time points. Fifty-one of the regulated genes were associated with apoptosis and were grouped into five clusters based on their expression patterns. Early in infection (6 and 12 h), genes with antiapoptotic roles were upregulated and genes with apoptotic roles were downregulated. Later in infection (24, 48, and 72 h), proapoptotic genes were induced and antiapoptotic genes were downregulated, suggesting a biphasic regulation of apoptosis: antiapoptotic state early and moderately proapoptotic state late in infection. This transcriptional profile matched the actual occurrence of apoptosis in the infected cultures. Apoptosis was first detected at 12 h postinfection and increased to a plateau at 24 h, when 20% of infected cells showed nuclear condensation. In contrast, experimental silencing of Bcl-2 induced apoptosis in 50% of infected cells at 12 h postinfection. This resulted in a decrease in the infection rate and a reduction in the accumulation of meront-containing cells. To test the significance of the moderately proapoptotic state late in the infection, we inhibited apoptosis using pancaspase inhibitor Z-VAD-FMK. This treatment also affected the progression of C. parvum infection, as reinfection, normally seen late (24 h to 48 h), did not occur and accumulation of mature meronts was impaired. Control of host apoptosis is complex and crucial to the life of C. parvum. Apoptosis control has at least two components, early inhibition and late moderate promotion. For a successful infection, both aspects appear to be required.

Cryptosporidium parvum is an obligate intracellular protozoan capable of causing severe diarrheal disease in a wide variety of mammals, including humans. C. parvum infection has been associated with induction of apoptosis in exposed epithelial cells, and we now demonstrate that apoptosis is restricted to a subset of cells actively infected with C. parvum. Approximately 20% of the infected cells underwent apoptosis within 48 h of infection, suggesting that the majority of the infected cells are rescued from apoptosis. C. parvum infection resulted in low-level activation of multiple members of the caspase family, including caspase-2, -3, -4, -6, -8, and -9. The kinetics of caspase activation correlated with apoptosis over a 48-h time course. Pan caspase inhibitors reduced apoptosis of epithelial cells infected by C. parvum. Furthermore, C. parvum infection inhibited staurosporine-induced apoptosis and caspase-3/7 activation at 24 h and 48 h. Infection with C. parvum led to upregulation of genes encoding inhibitors of apoptosis proteins (IAPs), including c-IAP1, c-IAP2, XIAP, and survivin. Knockdown of survivin gene expression, but not that of c-IAP1, c-IAP2, or XIAP expression, increased caspase-3/7 activity as well as apoptosis of infected cells and decreased C. parvum 18S rRNA levels. These data suggest that the apoptotic response of infected intestinal epithelial cells is actively suppressed by C. parvum via upregulation of survivin, favoring parasite infection.

The role of TNFalpha in regulating apoptotic signaling was investigated during subacute, low-dose (5.0 mg/kg) dimethylnitrosamine (DMN)-induced hepatotoxicity. In TNFalpha receptor (TNFR) intact (wild-type, WT) mice following 4 and 7 DMN exposures, hepatic transcripts for TNFalpha and TNFR-1 were elevated as compared to vehicle controls. DMN hepatotoxicity in WT and TNFR-1/TNFR-2 double knockout (DKO) mice were then compared over a 7-d exposure period. Liver RNA was isolated to measure hepatic expression of TNFalpha/Fas-related genes and the Bcl-2 family of genes that impact apoptosis. Hepatic mRNA levels for Fas, the apoptosis-promoting gene Bax, and the anti-apoptotic gene, Bcl-X(L), were up regulated following 4 and 7 DMN exposures in both WT and TNFR DKO mice as compared to vehicle controls. Notably, hepatic transcript levels for Bax were higher in TNFR DKO mice treated with DMN compared to identically treated WT mice. However, we detected approximately equal DMN-induced apoptotic degradation of liver DNA following 1, 4, and 7 exposures in WT and TNFR DKO mice. Taken together, these data show DMN-induced hepatic TNFalpha expression and suggest that TNFR-1 signaling may be up regulated following 4 and 7 daily DMN exposures. However, TNFalpha is not required for apoptotic signaling at the mRNA transcript level within the liver and instead may actually decrease Bax production.

Tumor necrosis factor receptor knockout (TNFR KO) mice were used to examine the role of tumor necrosis factor-alpha (TNFalpha) signaling during acute hepatotoxicant exposure. Mice were exposed intraperitoneally (ip) to either vehicle, phosphate-buffered saline (PBS), or dimethylnitrosamine (DMN, 100 mg/kg) for 24 h. Histological evaluation showed that DMN-treated TNFR-2 KO mice had increased liver damage compared to wild type (WT), TNFR-1 KO, or TNFR double KO (DKO) mice. Also, 3 of 8 TNFR-2 KO mice died following DMN treatment, suggesting that hepatic TNFR-2 signaling produces protective responses that counteract TNFR-1-mediated damage. DMN-induced cellular infiltration was absent in TNFR-1-deficient mice, indicating that infiltrating cells do not exacerbate acute hepatotoxic events. In separate experiments, mice were exposed ip to either DMN (5.0 or 100 mg/kg), carbon tetrachloride (CCl4, 0.3 or 1.0 ml/kg), or corresponding PBS/corn oil controls for 6 or 24 h to compare the hepatic mRNA expression of cytokine- and apoptotic-associated genes. Following 24 h of DMN (100 mg/kg) or 6-24 h of CCl4 treatment, hepatic transcripts for TNFalpha, interferon (IFN)-gamma, IL (interleukin)-1RI, and transforming growth factor (TGF)-betaRII were induced. Hepatotoxicant-treated WT and TNFR DKO mice induced liver transcripts for the pro- and anti-apoptotic genes, Bax and Bcl-X(L), respectively, indicating TNF-independent gene activation. The anti-apoptotic gene, Bfl-1, was highly expressed in CCl4-treated, TNFR-positive strains, but minimally expressed in TNFR DKO mice, suggesting that hepatic Bfl-1 is TNF-regulated. Taken together, these data show that acute hepatotoxicant exposure is followed by upregulation of liver cytokine, cytokine receptor, and apoptotic transcripts, and that TNFalpha regulates various aspects of liver inflammation and injury in a TNFR-specific fashion.