The inter-alpha-trypsin inhibitor (IαI) complex is composed of the bikunin core protein with a single chondroitin sulfate (CS) attached and one or two heavy chains (HCs) covalently linked to the CS chain. The HCs from IαI can be transferred to hyaluronan (HA) through a TNFα-stimulated gene-6 (TSG-6) dependent process to form an HC•HA matrix. Previous studies reported increased IαI, HA, and HC•HA complexes in mouse bronchoalveolar lavage fluid (BALF) post-influenza infection. However, the expression and incorporation of HCs into the HA matrix of the lungs during the clinical course of influenza A virus (IAV) infection and the biological significance of the HC•HA matrix are poorly understood. The present study aimed to better understand the composition of HC•HA matrices in mice infected with IAV and how these matrices regulate the host pulmonary immune response. In IAV infected mice bikunin, HC1–3, TSG-6, and HAS1–3 all show increased gene expression at various times during a 12-day clinical course. The increased accumulation of IαI and HA was confirmed in the lungs of infected mice using immunohistochemistry and quantitative digital pathology. Western blots confirmed increases in the IαI components in BALF and lung tissue at 6 days post-infection (dpi). Interestingly, HCs and bikunin recovered from BALF and plasma from mice 6 dpi with IAV, displayed differences in the HC composition by Western blot analysis and differences in bikunin's CS chain sulfation patterns by mass spectrometry analysis. This strongly suggests that the IαI components were synthesized in the lungs rather than translocated from the vascular compartment. HA was significantly increased in BALF at 6 dpi, and the HA recovered in BALF and lung tissues were modified with HCs indicating the presence of an HC•HA matrix. In vitro experiments using polyinosinic-polycytidylic acid (poly(I:C)) treated mouse lung fibroblasts (MLF) showed that modification of HA with HCs increased cell-associated HA, and that this increase was due to the retention of HA in the MLF glycocalyx. In vitro studies of leukocyte adhesion showed differential binding of lymphoid (Hut78), monocyte (U937), and neutrophil (dHL60) cell lines to HA and HC•HA matrices. Hut78 cells adhered to immobilized HA in a size and concentration-dependent manner. In contrast, the binding of dHL60 and U937 cells depended on generating a HC•HA matrix by MLF. Our in vivo findings, using multiple bronchoalveolar lavages, correlated with our in vitro findings in that lymphoid cells bound more tightly to the HA-glycocalyx in the lungs of influenza-infected mice than neutrophils and mononuclear phagocytes (MNPs). The neutrophils and MNPs were associated with a HC•HA matrix and were more readily lavaged from the lungs. In conclusion, this work shows increased IαI and HA accumulation and the formation of a HC•HA matrix in mouse lungs post-IAV infection. The formation of HA and HC•HA matrices could potentially create specific microenvironments in the lungs for immune cell recruitment and activation during IAV infection.

α-胰蛋白酶抑制剂间 （IαI） 复合物由连接单个硫酸软骨素 （CS） 的双核蛋白核心蛋白和一条或两条与 CS 链共价连接的重链 （HC） 组成。来自 IαI 的 HCs 可以通过 TNFα 刺激的基因 6 （TSG-6） 依赖性过程转移到透明质酸 （HA），形成 HC•HA 基质。先前的研究报道了流感感染后小鼠支气管肺泡灌洗液 （BALF） 中 IαI 、 HA 和 HC•HA 复合物的增加。然而，在甲型流感病毒 （IAV） 感染的临床过程中，HCs 的表达和掺入肺部的 HA 基质以及 HC•HA 基质的生物学意义知之甚少。本研究旨在更好地了解 IAV 感染小鼠中 HC•HA 基质的组成以及这些基质如何调节宿主肺免疫反应。在 IAV 感染的小鼠中，bikunin、HC1-3、TSG-6 和 HAS1-3 在 12 天的临床病程中均表现出不同时间的基因表达增加。使用免疫组化和定量数字病理学证实 IαI 和 HA 在感染小鼠肺部积累增加。Western 印迹证实感染后 6 天 （dpi） BALF 和肺组织中 IαI 成分增加。有趣的是，从 BALF 和使用 IAV 的小鼠血浆 6 dpi 中回收的 HCs 和 bikunin 通过 Western blot 分析显示 HC 组成的差异，通过质谱分析显示 bikunin 的 CS 链硫酸化模式的差异。这强烈表明 IαI 成分是在肺部合成的，而不是从血管隔室易位的。 HA 在 6 dpi 时在 BALF 中显著增加，在 BALF 和肺组织中回收的 HA 用 HCs 修饰，表明存在 HC•HA 基质。使用聚肌苷-聚胞苷酸 （poly（I：C）） 处理的小鼠肺成纤维细胞 （MLF） 的体外实验表明，用 HCs 修饰 HA 会增加细胞相关 HA，而这种增加是由于 HA 保留在 MLF 糖萼中。白细胞粘附的体外研究表明，淋巴细胞 （Hut78） 、单核细胞 （U937） 和中性粒细胞 （dHL60） 细胞系与 HA 和 HC•HA 基质的结合存在差异。Hut78 细胞以大小和浓度依赖性方式粘附在固定的 HA 上。相比之下，dHL60 和 U937 细胞的结合依赖于 MLF 生成 HC•HA 基质。我们使用多次支气管肺泡灌洗液的体内研究结果与我们的体外研究结果相关，即淋巴细胞与流感感染小鼠肺部的 HA 糖萼结合比中性粒细胞和单核吞噬细胞 （MNP） 更紧密。中性粒细胞和 MNP 与 HC•HA 基质相关，更容易从肺部灌洗。总之，这项工作显示 IAV 感染后小鼠肺中 IαI 和 HA 积累增加以及 HC•HA 基质的形成。HA 和 HC•HA 基质的形成可能会在肺部产生特定的微环境，以便在 IAV 感染期间为免疫细胞募集和激活。