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Cryptococcus neoformans induces IL-8 secretion and CXCL1 expression by human bronchial epithelial cells.

Guillot L, Carroll SF, Badawy M, Qureshi ST - Respir. Res. (2008)

Bottom Line: Viable C. neoformans is a strong activator of BEAS-2B cells, resulting in the production of the neutrophil chemokine Interleukin (IL)-8 in a time- and dose-dependent manner.NHBE was highly responsive to stimulation with C. neoformans; in addition to transcriptional up regulation of CXCL1, these primary cells exhibited the greatest IL-8 secretion and cell damage in response to stimulation with an acapsular strain of C. neoformans.The presence of capsular polysaccharide and in vitro fungal culture conditions modulate the host inflammatory response to C. neoformans.

View Article: PubMed Central - HTML - PubMed

Affiliation: McGill Centre for the Study of Host Resistance, Room L11-403, 1650 Cedar Avenue, Montreal, QC, H3G 1A4, Canada. loic.guillot@mail.mcgill.ca

ABSTRACT

Background: Cryptococcus neoformans (C. neoformans) is a globally distributed fungal pathogen with the potential to cause serious disease, particularly among immune compromised hosts. Exposure to this organism is believed to occur by inhalation and may result in pneumonia and/or disseminated infection of the brain as well as other organs. Little is known about the role of airway epithelial cells in cryptococcal recognition or their ability to induce an inflammatory response.

Methods: Immortalized BEAS-2B bronchial epithelial cells and primary normal human bronchial epithelium (NHBE) were stimulated in vitro with encapsulated or acapsular C. neoformans cultivated at room temperature or 37 degrees C. Activation of bronchial epithelial cells was characterized by analysis of inflammatory cytokine and chemokine expression, transcription factor activation, fungal-host cell association, and host cell damage.

Results: Viable C. neoformans is a strong activator of BEAS-2B cells, resulting in the production of the neutrophil chemokine Interleukin (IL)-8 in a time- and dose-dependent manner. IL-8 production was observed only in response to acapsular C. neoformans that was grown at 37 degrees C. C. neoformans was also able to induce the expression of the chemokine CXCL1 and the transcription factor CAAT/enhancer-binding protein beta (CEBP/beta) in BEAS-2B cells. NHBE was highly responsive to stimulation with C. neoformans; in addition to transcriptional up regulation of CXCL1, these primary cells exhibited the greatest IL-8 secretion and cell damage in response to stimulation with an acapsular strain of C. neoformans.

Conclusion: This study demonstrates that human bronchial epithelial cells mediate an acute inflammatory response to C. neoformans and are susceptible to damage by this fungal pathogen. The presence of capsular polysaccharide and in vitro fungal culture conditions modulate the host inflammatory response to C. neoformans. Human bronchial epithelial cells are likely to contribute to the initial stages of pulmonary host defense in vivo.

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NHBE cells induce chemokine expression and are susceptible to damage following stimulation with C. neoformans. (A) NHBE cells were left unstimulated (NS, white box) or stimulated with a MOI = 20 of capsular or acapsular C. neoformans cultured at RT or 37°C, as indicated. Twenty-four hours later, supernatants were collected and ELISA was used to measure IL-8 concentrations. Results are expressed as the mean ± S.D. of triplicate measurements and are representative of three independent experiments. (B) Supernatants were also assayed for LDH release using colorimetry. Data are expressed as a percentage of LDH release from unstimulated cells treated with a detergent lysis solution (triton) and represent the mean ± S.D. of triplicate measurements from two independent experiments. (C, D) Relative quantification of CXCL1 and CEBP/β expression was determined by real time PCR. Results are representative of the mean ± S.D. of one experiment performed in triplicate; δ P ≤ 0.05 vs. NS, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
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Figure 6: NHBE cells induce chemokine expression and are susceptible to damage following stimulation with C. neoformans. (A) NHBE cells were left unstimulated (NS, white box) or stimulated with a MOI = 20 of capsular or acapsular C. neoformans cultured at RT or 37°C, as indicated. Twenty-four hours later, supernatants were collected and ELISA was used to measure IL-8 concentrations. Results are expressed as the mean ± S.D. of triplicate measurements and are representative of three independent experiments. (B) Supernatants were also assayed for LDH release using colorimetry. Data are expressed as a percentage of LDH release from unstimulated cells treated with a detergent lysis solution (triton) and represent the mean ± S.D. of triplicate measurements from two independent experiments. (C, D) Relative quantification of CXCL1 and CEBP/β expression was determined by real time PCR. Results are representative of the mean ± S.D. of one experiment performed in triplicate; δ P ≤ 0.05 vs. NS, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.

Mentions: To further characterize the activation profile of bronchial epithelial cells by C. neoformans, we used an oligonucleotide microarray in order to examine the induction of a panel of 113 inflammatory cytokines, chemokines, and their receptors (a complete list of genes represented on the microarray is available on request). In unstimulated cells, we observed constitutive expression of C3, C4A, CXCL-10, MIF and TNFR1A that was not significantly influenced by C. neoformans stimulation (Figure 5A). Twenty-four hours after stimulation with acapsular C. neoformans, we observed the induction of IL-8, as expected, as well as up-regulation of CCL2, CXCL1, CCL15, and CEBP/β (Figure 5A and 5B). To validate these observations, we analyzed the expression of CXCL1, CCL2, and CEBP/β, and CCL15 by qualitative and real-time PCR. As shown in Figures 6C and 6D, we confirmed modest up-regulation of CXCL-1 and CEBP/β, but not CCL2, in C. neoformans stimulated cells compared to untreated BEAS-2B cells. Surprisingly, we were not able to demonstrate the expression of CCL15 either in unstimulated or C. neoformans-stimulated BEAS-2B cells by RT-PCR; nevertheless, we were able to weakly amplify CCL15 using a human reference cDNA as a positive control (kindly provided by SuperArray; data not shown). The integrity of all RNA preparations was verified by RT-PCR analysis of β-actin expression.


Cryptococcus neoformans induces IL-8 secretion and CXCL1 expression by human bronchial epithelial cells.

Guillot L, Carroll SF, Badawy M, Qureshi ST - Respir. Res. (2008)

NHBE cells induce chemokine expression and are susceptible to damage following stimulation with C. neoformans. (A) NHBE cells were left unstimulated (NS, white box) or stimulated with a MOI = 20 of capsular or acapsular C. neoformans cultured at RT or 37°C, as indicated. Twenty-four hours later, supernatants were collected and ELISA was used to measure IL-8 concentrations. Results are expressed as the mean ± S.D. of triplicate measurements and are representative of three independent experiments. (B) Supernatants were also assayed for LDH release using colorimetry. Data are expressed as a percentage of LDH release from unstimulated cells treated with a detergent lysis solution (triton) and represent the mean ± S.D. of triplicate measurements from two independent experiments. (C, D) Relative quantification of CXCL1 and CEBP/β expression was determined by real time PCR. Results are representative of the mean ± S.D. of one experiment performed in triplicate; δ P ≤ 0.05 vs. NS, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
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Related In: Results  -  Collection

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Figure 6: NHBE cells induce chemokine expression and are susceptible to damage following stimulation with C. neoformans. (A) NHBE cells were left unstimulated (NS, white box) or stimulated with a MOI = 20 of capsular or acapsular C. neoformans cultured at RT or 37°C, as indicated. Twenty-four hours later, supernatants were collected and ELISA was used to measure IL-8 concentrations. Results are expressed as the mean ± S.D. of triplicate measurements and are representative of three independent experiments. (B) Supernatants were also assayed for LDH release using colorimetry. Data are expressed as a percentage of LDH release from unstimulated cells treated with a detergent lysis solution (triton) and represent the mean ± S.D. of triplicate measurements from two independent experiments. (C, D) Relative quantification of CXCL1 and CEBP/β expression was determined by real time PCR. Results are representative of the mean ± S.D. of one experiment performed in triplicate; δ P ≤ 0.05 vs. NS, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001.
Mentions: To further characterize the activation profile of bronchial epithelial cells by C. neoformans, we used an oligonucleotide microarray in order to examine the induction of a panel of 113 inflammatory cytokines, chemokines, and their receptors (a complete list of genes represented on the microarray is available on request). In unstimulated cells, we observed constitutive expression of C3, C4A, CXCL-10, MIF and TNFR1A that was not significantly influenced by C. neoformans stimulation (Figure 5A). Twenty-four hours after stimulation with acapsular C. neoformans, we observed the induction of IL-8, as expected, as well as up-regulation of CCL2, CXCL1, CCL15, and CEBP/β (Figure 5A and 5B). To validate these observations, we analyzed the expression of CXCL1, CCL2, and CEBP/β, and CCL15 by qualitative and real-time PCR. As shown in Figures 6C and 6D, we confirmed modest up-regulation of CXCL-1 and CEBP/β, but not CCL2, in C. neoformans stimulated cells compared to untreated BEAS-2B cells. Surprisingly, we were not able to demonstrate the expression of CCL15 either in unstimulated or C. neoformans-stimulated BEAS-2B cells by RT-PCR; nevertheless, we were able to weakly amplify CCL15 using a human reference cDNA as a positive control (kindly provided by SuperArray; data not shown). The integrity of all RNA preparations was verified by RT-PCR analysis of β-actin expression.

Bottom Line: Viable C. neoformans is a strong activator of BEAS-2B cells, resulting in the production of the neutrophil chemokine Interleukin (IL)-8 in a time- and dose-dependent manner.NHBE was highly responsive to stimulation with C. neoformans; in addition to transcriptional up regulation of CXCL1, these primary cells exhibited the greatest IL-8 secretion and cell damage in response to stimulation with an acapsular strain of C. neoformans.The presence of capsular polysaccharide and in vitro fungal culture conditions modulate the host inflammatory response to C. neoformans.

View Article: PubMed Central - HTML - PubMed

Affiliation: McGill Centre for the Study of Host Resistance, Room L11-403, 1650 Cedar Avenue, Montreal, QC, H3G 1A4, Canada. loic.guillot@mail.mcgill.ca

ABSTRACT

Background: Cryptococcus neoformans (C. neoformans) is a globally distributed fungal pathogen with the potential to cause serious disease, particularly among immune compromised hosts. Exposure to this organism is believed to occur by inhalation and may result in pneumonia and/or disseminated infection of the brain as well as other organs. Little is known about the role of airway epithelial cells in cryptococcal recognition or their ability to induce an inflammatory response.

Methods: Immortalized BEAS-2B bronchial epithelial cells and primary normal human bronchial epithelium (NHBE) were stimulated in vitro with encapsulated or acapsular C. neoformans cultivated at room temperature or 37 degrees C. Activation of bronchial epithelial cells was characterized by analysis of inflammatory cytokine and chemokine expression, transcription factor activation, fungal-host cell association, and host cell damage.

Results: Viable C. neoformans is a strong activator of BEAS-2B cells, resulting in the production of the neutrophil chemokine Interleukin (IL)-8 in a time- and dose-dependent manner. IL-8 production was observed only in response to acapsular C. neoformans that was grown at 37 degrees C. C. neoformans was also able to induce the expression of the chemokine CXCL1 and the transcription factor CAAT/enhancer-binding protein beta (CEBP/beta) in BEAS-2B cells. NHBE was highly responsive to stimulation with C. neoformans; in addition to transcriptional up regulation of CXCL1, these primary cells exhibited the greatest IL-8 secretion and cell damage in response to stimulation with an acapsular strain of C. neoformans.

Conclusion: This study demonstrates that human bronchial epithelial cells mediate an acute inflammatory response to C. neoformans and are susceptible to damage by this fungal pathogen. The presence of capsular polysaccharide and in vitro fungal culture conditions modulate the host inflammatory response to C. neoformans. Human bronchial epithelial cells are likely to contribute to the initial stages of pulmonary host defense in vivo.

Show MeSH
Related in: MedlinePlus