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TLR4 promotes Cryptosporidium parvum clearance in a mouse model of biliary cryptosporidiosis - PubMed

TLR4 promotes Cryptosporidium parvum clearance in a mouse model of biliary cryptosporidiosis

Steven P O'Hara et al. J Parasitol. 2011 Oct.

Abstract

Cholangiocytes, the epithelial cells lining intrahepatic bile ducts, express multiple toll-like receptors (TLRs) and, thus, have the capacity to recognize and respond to microbial pathogens. In previous work, we demonstrated that TLR4, which is activated by gram-negative lipopolysaccharide (LPS), is upregulated in cholangiocytes in response to infection with Cryptosporidium parvum in vitro and contributes to nuclear factor-kappaB (NF-kB) activation. Here, using an in vivo model of biliary cryptosporidiosis, we addressed the functional role of TLR4 in C. parvum infection dynamics and hepatobiliary pathophysiology. We observed that C57BL mice clear the infection by 3 wk post-infection (PI). In contrast, parasites were detected in bile and stool in TLR4-deficient mice at 4 wk PI. The liver enzymes alanine transaminase (ALT) and aspartate transaminase (AST), and the proinflammatory cytokines tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-6 peaked at 1 to 2 wk PI and normalized by 4 wk in infected C57BL mice. C57BL mice also demonstrated increased cholangiocyte proliferation (PCNA staining) at 1 wk PI that was resolved by 2 wk PI. In contrast, TLR4-deficient mice showed persistently elevated serum ALT and AST, elevated hepatic IL-6 levels, and histological evidence of hepatocyte necrosis, increased inflammatory cell infiltration, and cholangiocyte proliferation through 4 wk PI. These data suggest that a TLR4-mediated response is required for efficient eradication of biliary C. parvum infection in vivo, and lack of this pattern-recognition receptor contributes to an altered inflammatory response and an increase in hepatobiliary pathology.

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Figures

Figure 1
Figure 1

TLR4 knock-out mice are less efficient at eradicating C. parvum biliary infection. 106 C. parvum oocysts were injected into the gallbladder of wild type C57BL or TLR4 knock-out mice. A. C. parvum infection in the intrahepatic ducts of wild type mice was detected by H&E staining one week post-injection. B. More parasites were found in the intrahepatic ducts of TLR4 knock-out mice one week post-infection compared with wild type. C. C. parvum parasites were not found in the intrahepatic bile ducts in the wild type mice four weeks post-infection, while parasites were detected in TLR4 knock-out mice (D). Insets in A – D are higher power of the boxed areas showing the parasite (arrowheads). E. Quantitative analysis showing parasite number in the intrahepatic bile ducts in the wild type and TLR4 knock-out mice one and four weeks post-infection. F. WT and TLR4 KO stool samples were stained with carbol fuscin and analyzed by light microscopy. Wild type mice showed a greater number of oocysts shed in the stool at week 1 post-inoculation with a significant drop at week 2. By weeks 3 and 4 post-injection, oocysts were undetected indicating clearance of the C. parvum oocyst by the wild type mice. Conversely, oocyst shedding was detected in knock out mice from week 1 to week 4 post-inoculation indicating persistence of C. parvum infection. *, p < 0.05; **, p < 0.01 compared to wild type. *, P < 0.05 compared with wild type mice.

Figure 2
Figure 2

Immunofluorescent detection of C. parvum in the bile of infected mice. Bile was collected from infected mice. 50 µl of bile was cytospun onto glass slides, and immunofluorescence was performed to detect the parasite (A, B). A. Multiple CP2 antibody positive parasites were detected in the bile of both wild type C57BL mice (A) and TLR4-deficient mice (B) one week post-infection, Bars = 10 µm. C. Quantification of parasites observed over the course of 4 weeks. The slides containing the immunofluorescently labeled parasites were used to quantify the number of parasites present in bile. Fifteen 40X fields were observed from each slide, and the number of parasites per 40X field (presented as mean ± SEM) was calculated. By 2 weeks post-infection, the number of parasites observed in the bile of B57BL mice drops precipitously and by 3 weeks post-infection, no parasites were observed. In contrast, while the numbers of parasites are decreased, parasites were detected in the bile of TLR4 deficient mice through 4 weeks post-infection. *, p < 0.05 compared to wild type.

Figure 3
Figure 3

Analysis of proinflammatory cytokine expression (ELISA) in homogenized livers of C. parvum infected wild type (solid lines) and TLR4 deficient (dashed lines) C57BL mice. A. IFNγ expression in wild type mice peaked at one week post-infection and was significantly greater than control uninfected mice (*, p<0.05) and TLR4 deficient mice (#, p<0.05). The expression of IFNγ in TLR4 deficient mice was not significantly increased compared to matched, uninfected controls (0 wks) at any time point. B. TNFα expression in wild type C57BL mice peaked at 2 weeks post-infection and was significantly different than matched, uninfected controls (*, p<0.05). The expression of TNFα in TLR4 deficient mice was significantly greater than matched uninfected controls at 3 and 4 weeks post-infection (*, p<0.05). Expression of TNFα was not significantly different between the wild type and TLR4 deficient C57BL mice at any time point. C. IL6 expression in wild type mice peaked at one week post-infection and was significantly increased compared to matched uninfected controls at this time point (*, p<0.05). In contrast, IL-6 expression in TLR4 deficient mice was significantly increased compared to matched uninfected controls (0 wks) at each time point (*, p<0.05). Furthermore, IL-6 expression was significantly increased compared to wild type C57BL mice at weeks 1, 3, and 4 (#, p<0.05). D. IL-6 expression was measured by ELISA in cultured human cholangiocytes (H69, solid line) and H69 cells stably expressing the TLR4-DN (dashed line). IL-6 expression peaked at 4 hrs post-infection in the H69 cells and was significantly elevated compared to uninfected controls (*, p<0.05) and cells expressing the TLR4-DN (#, p<0.05) and diminished by 12 hrs post-infection. IL-6 expression in cells expressing the TLR4-DN peaked at 12 hr post-infection and was significantly elevated compared to matched uninfected controls (*, p<0.01) and H69 cells (#, p<0.01) at 12 and 24 hr post-infection. Values are presented as mean +/− SE.

Figure 4
Figure 4

Liver enzyme serum biochemistries. Serum was obtained from C. parvum infected wild type and TLR4 deficient C57BL mice. A. Serum alkaline phosphatase (Alk Phos) levels from wild type C57BL mice were not significantly elevated at any time point compared to uninfected controls (0 wks). In contrast, the serum alkaline phosphatase level of C. parvum infected TLR4 deficient mice was significantly elevated compared to matched uninfected controls at one week post-infection. B. Serum Aspartate transaminase levels peaked at 2 weeks post-infection for both wild type and TLR4 deficient C57BL mice, reaching significance only for the TLR4 deficient mice (*, p<0.05). AST levels diminish in wild type C57BL mice by 3 weeks post-infection, but remain significantly elevated in the TLR4 deficient mice compared to matched, uninfected controls at 3 and 4 weeks post-infection (*, p<0.05). In addition, the levels of AST were significantly elevated compared to infected wild type mice at 4 weeks post-infection (#, p<0.05). C. Serum Alanine transaminase levels again peaked at 2 weeks post-infection for both wild type and TLR4 deficient C57BL mice and were significantly elevated compared to matched, uninfected controls (p<0.05). ALT levels diminished to levels comparable to uninfected, matched controls by week three post-infection. In contrast, C. parvum infected TLR4 deficient mice exhibited elevated levels of ALT at weeks 3 and 4 post-infection compared to matched uninfected controls (*p<0.05) and wild type infected mice (#, p<0.05). Values are presented as mean +/− SE.

Figure 5
Figure 5

Hematoxylin and Eosin staining of C. parvum infected wild type and TLR4 deficient C57BL mouse livers. Top panels show typical staining of wild type mouse livers from week one to four post-infection. A. At week 1 post infection the livers typically exhibit minimal to moderate inflammation, with the infiltration of polymorphonucleocytes. Inflammatory cell infiltration remains mild to moderate through weeks 2 and 3 with some cholangitis and grade 2 ductular proliferation. B. By week 4 post-infection inflammation is reduced to minimal with normal histology. C. In contrast, H&E staining of TLR4 deficient mouse livers at 1 week post-infection demonstrates moderate to severe inflammation with multifocal necrosis and grade 3 ductular proliferation. By week two post-infection mild to moderate inflammation was observed in the portal tracts of TLR4 deficient mice. D. By week four post-infection, severe portal tract inflammation with hepatocellular necrosis and grade 2–3 ductular proliferation was observed.

Figure 6
Figure 6

Cholangiocyte proliferation was assessed by PCNA staining of C. parvum infected wild type (top panels) and TLR4 deficient (bottom panels) mice. A. Wild type C57BL mice exhibited minimal ductular proliferation at week 1. B. By week four post-infection the levels of proliferation decreased to undetectable levels in wild-type mice. C. parvum infected TLR4 deficient mouse livers exhibited increased levels of cholangiocyte proliferation at I week post-infection (C) through week four post-infection (D). E. The level of cholangiocyte proliferation was quantified by counting the number of PCNA positive cells per bile duct from a minimum of 10 ducts and 3 mice for each group (30 ducts minimum). TLR4 deficient mice exhibited increased PCNA staining compared to wild-type controls at both 1 week and 4 weeks post-infection. *, p<0.01 compared to wild type at same time point.

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