Thoracic Diseases Research Unit, Departments of Medicine and Biochemistry, Mayo Clinic College of Medicine, Rochester, Minnesota, 55905, USA.
J Med Microbiol. 2023 Jun;72(6). doi: 10.1099/jmm.0.001714.
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 .
C 型凝集素受体 (CLRs) 在髓样细胞上表达丰富，在那里它们发挥多种功能，包括作为模式识别受体 (PRRs) 来驱动病原体的固有和适应性免疫。根据酪氨酸基信号基序的存在，CLR-微生物病原体的相互作用可能导致抗炎或促炎信号。在本手稿中，我们报告了我们实验室对两种识别细胞壁同源物 (CWH) 和纯化细胞壁级分 (CWF) 的新型 CLR 的研究。为了研究新生成的 hFc-CLR 融合物在与 CWHs 和 CWFs 结合以及随后的下游炎症信号分析中的潜力。通过改良 ELISA 筛选新生成的 hFc-CLR 融合物 CLEC4A 和 CLEC12B 与 CWHs 和 CWFs 制剂。免疫荧光分析 (IFA) 用于可视化 hFc-CLR 融合物与完整固定真菌生活形式的结合，以验证结果。采用定量 PCR (q-PCR) 分析免疫抑制性肺孢子菌肺炎 (PCP) 模型小鼠与未感染小鼠的肺 mRNA，以检测各自和基因转录本的可能变化。最后，使用 siRNA 技术对两种 CLR 进行处理，以确定在存在 CWFs 的情况下刺激小鼠巨噬细胞时对下游炎症事件的影响。我们确定 CLEC4A 和 CLEC12B 两种 hFc-CLR 均与 CWHs 和 CWFs 显示出显著结合。结合事件显示与壳聚糖和昆布多糖均有显著结合，这两种多糖均含有β-(1,3)葡聚糖以及乙酰葡萄糖胺 (GlcNAc) 残基，并且与阴性对照碳水化合物右旋糖轻微但无显著结合。IFA 用两种 CLR hFc 融合物对整个生活形式进行验证，证实了这些发现。最后，我们在免疫抑制性肺孢子菌肺炎 (PCP) 模型小鼠中检测了上述两种 CLR 的 mRNA 表达谱，并确定两种 CLR 在感染期间均显著上调。最后，对小鼠 RAW 巨噬细胞系中的两种 CLR 进行了 siRNA 处理，结果表明，在 CWF 刺激的巨噬细胞中，沉默对 TNF-α生成没有显著影响。相反，沉默 CLEC12B CLR 导致用相同 CWF 刺激的 RAW 细胞中 TNF-α的显著减少。这里提供的资料为识别的 CLR 家族的新成员提供了信息。在 PCP 小鼠模型中使用 CLEC4A 和/或 CLEC12B 缺陷小鼠进行的进一步研究应能进一步了解宿主对的免疫反应。
