1887

Abstract

C-type lectin receptors (CLRs) are prominently expressed on myeloid cells where they perform multiple functions including serving as pattern recognition receptors (PRRs) to drive innate as well as adaptive immunity to pathogens. Depending on the presence of a tyrosine-based signalling motif, CLR-microbial pathogen engagement may result in either anti- or pro-inflammatory signalling.

In this manuscript, we report our laboratory study of two novel CLRs that recognize cell wall homogenates (CWH) and a purified cell wall fraction (CWF).

To study the potential of newly generated hFc-CLR fusions on binding to CWHs and CWFs and subsequent downstream inflammatory signalling analysis.

Newly generated hFc-CLR fusion CLEC4A and CLEC12B were screened against CWHs and CWFs preparations via modified ELISA. Immunofluorescence assay (IFA) was utilized to visualize hFc-CLR fusion binding against intact fixed fungal life forms to verify results. Quantitative PCR (q-PCR) analysis of lung mRNA from the mouse immunosuppressed Pneumocystis pneumonia (PCP) model versus uninfected mice was employed to detect possible changes in the respective and transcripts. Lastly, siRNA technology of both CLRs was conducted to determine effects on downstream inflammatory events in mouse macrophages stimulated in the presence of CWFs.

We determined that both CLEC4A and CLEC12B hFc-CLRs displayed significant binding with CWHs and CWFs. Binding events showed significant binding to both curdlan and laminarin, both polysaccharides containing β-(1,3) glucans as well as -acetylglucosamine (GlcNAc) residues and modest yet non-significant binding to the negative control carbohydrate dextran. IFA with both CLR hFc-fusions against whole life forms corroborated these findings. Lastly, we surveyed the mRNA expression profiles of both CLRs tested above in the mouse immunosuppressed Pneumocystis pneumonia (PCP) model and determined that both CLRs were significantly up regulated during infection. Lastly, siRNA of both CLRs in the mouse RAW macrophage cell line was conducted and results demonstrated that silencing of resulted in no significant changes in TNF-alpha generation in CWF stimulated macrophages. On the contrary, silencing of CLR resulted in significant decreases in TNF-alpha in RAW cells stimulated with the same CWF.

The data presented here provide new members of the CLRs family recognizing . Future studies using CLEC4A and/or CLEC12B deficient mice in the PCP mouse model should provide further insights into the host immunological response to .

Funding
This study was supported by the:
  • National Institutes of Health (Award R01-HL62150)
    • Principle Award Recipient: AndrewH. Limper
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/content/journal/jmm/10.1099/jmm.0.001714
2023-06-09
2024-12-09
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