The extracellular matrix glycosaminoglycan, hyaluronan, has been described as a regulator of tissue inflammation, with hyaluronan fragments reported to stimulate innate immune cells. High molecular mass hyaluronan is normally present in tissues, but upon inflammation lower molecular mass fragments are generated. It is unclear if these hyaluronan fragments induce an inflammatory response or are a consequence of inflammation. In this study, mouse bone marrow derived macrophages and dendritic cells (DCs) were stimulated with various sizes of hyaluronan from different sources, fragmented hyaluronan, hyaluronidases and heavy chain modified-hyaluronan (HA-HC). Key pro-inflammatory molecules, tumour necrosis factor alpha, interleukin-1 beta, interleukin-12, CCL3, and the co-stimulatory molecules, CD40 and CD86 were measured. Only human umbilical cord hyaluronan, bovine testes and Streptomyces hyaluronlyticus hyaluronidase stimulated macrophages and DCs, however, these reagents were found to be contaminated with endotoxin, which was not fully removed by polymyxin B treatment. In contrast, pharmaceutical grade hyaluronan and hyaluronan fragments failed to stimulate in vitro-derived or ex vivo macrophages and DCs, and did not induce leukocyte recruitment after intratracheal instillation into mouse lungs. Hence, endotoxin-free pharmaceutical grade hyaluronan does not stimulate macrophages and DCs in our inflammatory models. These results emphasize the importance of ensuring hyaluronan preparations are endotoxin free.
Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.
Macrophages and dendritic cells (DCs) are innate immune cells found in tissues and lymphoid organs that play a key role in the defense against pathogens. However, they are difficult to isolate in sufficient numbers to study them in detail, therefore, in vitro models have been developed. In vitro cultures of bone marrow-derived macrophages and dendritic cells are well-established and valuable methods for immunological studies. Here, a method for culturing and identifying both DCs and macrophages from a single culture of primary mouse bone marrow cells using the cytokine granulocyte macrophage colony-stimulating factor (GM-CSF) is described. This protocol is based on the established procedure first developed by Lutz et al. in 1999 for bone marrow-derived DCs. The culture is heterogeneous, and MHCII and fluoresceinated hyaluronan (FL-HA) are used to distinguish macrophages from immature and mature DCs. These GM-CSF derived macrophages provide a convenient source of in vitro derived macrophages that closely resemble alveolar macrophages in both phenotype and function.
Recent evidence indicates that viral components of the microbiota can contribute to intestinal homeostasis and protection from local inflammatory or infectious insults. However, host-derived mechanisms that regulate the virome remain largely unknown. Here, we use colonization with the model commensal murine norovirus (MNV CR6) to interrogate host-directed mechanisms of viral regulation, and show that STAT1 is a central coordinator of both viral replication and antiviral T cell responses. In addition to restricting CR6 replication to the intestinal tract, we show that STAT1 regulates antiviral CD4 + and CD8 + T cell responses, and prevents systemic viral-induced tissue damage and disease. Despite altered T cell responses that resemble those that mediate lethal immunopathology in systemic viral infections in STAT1-deficient mice, depletion of adaptive immune cells and their associated effector functions had no effect on CR6-induced disease. However, therapeutic administration of an antiviral compound limited viral replication, preventing viral-induced tissue damage and death without impacting the generation of inflammatory antiviral T cell responses. Collectively, our data show that STAT1 restricts MNV CR6 replication within the intestinal mucosa, and that uncontrolled viral replication mediates disease rather than the concomitant development of dysregulated antiviral T cell responses in STAT1-deficient mice. Importance The intestinal microbiota is a collection of bacteria, archaea, fungi and viruses that colonize the mammalian gut. Co-evolution of the host and microbiota has required development of immunological tolerance to prevent ongoing inflammatory responses against intestinal microbes. Breakdown of tolerance to bacterial components of the microbiota can contribute to immune activation and inflammatory disease. However, the mechanisms that are necessary to maintain tolerance to viral components of the microbiome, and the consequences of loss of tolerance, are less well understood. Here, we show that STAT1 is integral for preventing escape of a commensal-like virus, murine norovirus CR6 (MNV CR6) from the gut, and that in the absence of STAT1, mice succumb to infection-induced disease. In contrast to other systemic viral infections, mortality of STAT1-deficient mice is not driven by immune-mediated pathology. Our data demonstrates the importance of host-mediated geographical restriction of commensal-like viruses.
The diverse commensal ecosystem present in the mammalian intestine requires the host immune system to maintain a tolerogenic environment. The virome is an understudied component of the microbiota which promotes intestinal homeostasis and protection from injury, but the host mechanisms that regulate tolerance to viral commensals are poorly described. The antiviral signal transducer STAT1 has previously been shown to mediate tolerance to systemic viral pathogens by suppressing adaptive immune responses and protecting the host from immunopathology, but we sought to characterize its role in the context of a commensal intestinal viral infection. We used a persistent strain of murine norovirus (MNV CR6) to interrogate host mechanisms of viral tolerance and commensalism, as CR6 has previously been shown to promote intestinal homeostasis. While CR6 infections of wildtype mice were asymptomatic and limited to the colon, STAT1-deficient mice exhibited virus-induced weight loss and mortality accompanied by systemic viral spread, colonic bacterial dysbiosis, CD4+ T cell dysfunction and hyperaccumulation of CD8+ T cells. However, clinical manifestation of virus-induced disease in STAT1-deficient mice was independent of T cells and the bacterial microbiota. Instead, therapeutic control of viral replication was sufficient to prevent virus-induced disease despite ongoing T cell dysregulation. Collectively, our data indicate that STAT1 maintains tolerance to a viral component of the intestinal microbiota by control of viral replication rather than immunopathology, suggesting that STAT1 uses distinct strategies to tolerate pathogenic and commensal viruses.
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