Effect of Culture Supernatant Derived from Trichophyton Rubrum Grown in the Nail Medium on the Innate Immunity-related Molecules of HaCaT / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Trichophyton rubrum is superficial fungi characteristically confined to dead keratinized tissues. These observations suggest that the soluble components released by the fungus could influence the host immune response in a cell in contact-free manner. Therefore, this research aimed to analyze whether the culture supernatant derived from T. rubrum grown in the nail medium could elicit the immune response of keratinocyte effectively.</p><p><b>METHODS</b>The culture supernatants of two strains (T1a, T XHB ) were compared for the β-glucan concentrations and their capacity to impact the innate immunity of keratinocytes. The β-glucan concentrations in the supernatants were determined with the fungal G-test kit and protein concentrations with bicinchoninic acid protein quantitative method, then HaCaT was stimulated with different concentrations of culture supernatants by adopting morphological method to select a suitable dosage. Expressions of host defense genes were assessed by quantitative polymerase chain reaction after the HaCaT was stimulated with the culture supernatants. Data were analyzed with one-way analysis of variance, followed by the least significant difference test.</p><p><b>RESULTS</b>The T. rubrum strains (T1a and T XHB ) released β-glucan of 87.530 ± 37.581 pg/ml and 15.747 ± 6.453 pg/ml, respectively into the media. The messenger RNA (mRNA) expressions of toll-like receptor-2 (TLR2), TLR4, and CARD9 were moderately up-regulated in HaCaT within 6-h applications of both supernatants. HaCaT cells were more responsive to T1a than T XHB . The slight increase of dendritic cells-specific intercellular adhesion molecule 3-grabbing nonintegrin expression was faster and stronger, induced by T1a supernatant than T XHB . The moderate decreases of RNase 7, the slight up-regulations of Dectin-1 and interleukin-8 at the mRNA level were detected only in response to T1a rather than T XHB . After a long-time contact, all the elevated defense genes decreased after 24 h.</p><p><b>CONCLUSION</b>The culture supernatant of T. rubrum could directly and transiently activate the innate immune response of keratinocytes.</p>

Exposure to heat-inactivated Trichophyton rubrum resulting in a limited immune response of human keratinocytes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Trichophyton rubrum (T. rubrum) represents the most important agent of dermatophytosis in humans. T. rubrum infection causes slight inflammation, and tends to be chronic and recurrent. It is suggested that it may result from the failure of epithelial cells to recognize T. rubrum effectively and initiate effective immune responses. The C-type lectin receptors (CLR) and toll-like receptors (TLR) are the two major pattern recognition receptors (PRRs) that recognize fungal components. Therefore, the purpose of the study was to analyze the expression of those PRRs and the cytokines in HaCaT cells stimulated with heat-inactivated T. rubrum conidia and hyphae, respectively.</p><p><b>METHODS</b>HaCaT cells were unstimulated or stimulated with heat-inactivated T. rubrum conidia and hyphae (1×10(6) and 1.5×10(5) colony-forming unit (CFU) in 2 ml medium, respectively) for 6, 12 and 24 hours. The mRNA expression of PRRs involved in recognizing fungal pathogen-associated molecular patterns (PAMPs) and signaling molecules were measured by quantitative reverse transcription polymerase chain reaction (RT-PCR). Meanwhile, surface toll-like receptor (TLR) 2, TLR4 and Dectin-1 were analyzed by fluorescence-activated cell sorter (FACS) 24 hours after treatment. The cytokines were detected in cell culture supernatants of HaCaT cells in 12 and 24 hours after treatment.</p><p><b>RESULTS</b>HaCaT cells constitutively expressed mRNA of membrane-bound TLR1, 2, 4 and 6, Dectin1 and DC-SIGN, but not Dectin-2 or Mincle. Heat-killed T. rubrum did not significantly upregulate gene transcriptions of the PRRs of HaCaT cells. Heat-inactivated T. rubrum conidia significantly reduced the surface expression of TLR2 and Dectin-1, and suppressed the secretions of interferon-inducible protein-10 (IP-10) and monocyte chemotactic protein-1 (MCP-1) of HaCaT cells, while heat-killed T. rubrum hyphae significantly induced the secretions of IP-10 and MCP-1.</p><p><b>CONCLUSION</b>The cell-wall antigens of T. rubrum fail to activate transcriptional expression of PRRs and induce a lower immune response of HaCaT cells by limited cytokines secretion.</p>