CD44 Loss Disrupts Lung Lipid Surfactant Homeostasis and Exacerbates Oxidized Lipid-Induced Lung Inflammation.

Alveolar macrophages (AMs) are CD44 expressing cells that reside in the alveolar space where they maintain lung homeostasis by serving critical roles in immunosurveillance and lipid surfactant catabolism. AMs lacking CD44 are unable to bind the glycosaminoglycan, hyaluronan, which compromises their survival and leads to reduced numbers of AMs in the lung. Using RNA sequencing, lipidomics and multiparameter flow cytometry, we demonstrate that CD44-/- mice have impaired AM lipid homeostasis and increased surfactant lipids in the lung. CD44-/- AMs had increased expression of CD36, a lipid scavenger receptor, as well as increased intracellular lipid droplets, giving them a foamy appearance. RNA sequencing revealed the differential expression of genes associated with lipid efflux and metabolism in CD44-/- AMs. Lipidomic analysis showed increased lipids in both the supernatant and cell pellet extracted from the bronchoalveolar lavage of CD44-/- mice. Phosphatidylcholine species, cholesterol, oxidized phospholipids and levels of reactive oxygen species (ROS) were increased in CD44-/- AMs. Oxidized phospholipids were more cytotoxic to CD44-/- AMs and induced greater lung inflammation in CD44-/- mice. Reconstitution of CD44+/+ mice with CD44-/- bone marrow as well as adoptive transfer of CD44-/- AMs into CD44+/+ mice showed that lipid accumulation in CD44-/- AMs occurred irrespective of the lung environment, suggesting a cell intrinsic defect. Administration of colony stimulating factor 2 (CSF-2), a critical factor in AM development and maintenance, increased AM numbers in CD44-/- mice and decreased phosphatidylcholine levels in the bronchoalveolar lavage, but was unable to decrease intracellular lipid accumulation in CD44-/- AMs. Peroxisome proliferator-activated receptor gamma (PPARγ), downstream of CSF-2 signaling and a regulator of lipid metabolism, was reduced in the nucleus of CD44-/- AMs, and PPARγ inhibition in normal AMs increased their lipid droplets. Thus, CD44 deficiency causes defects in AMs that lead to abnormal lipid accumulation and oxidation, which exacerbates oxidized lipid-induced lung inflammation. Collectively, these findings implicate CD44 as a regulator of lung homeostasis and inflammation.

Hyaluronan and Its Interactions With Immune Cells in the Healthy and Inflamed Lung.

Hyaluronan is a hygroscopic glycosaminoglycan that contributes to both extracellular and pericellular matrices. While the production of hyaluronan is essential for mammalian development, less is known about its interaction and function with immune cells. Here we review what is known about hyaluronan in the lung and how it impacts immune cells, both at homeostasis and during lung inflammation and fibrosis. In the healthy lung, alveolar macrophages provide the first line of defense and play important roles in immunosurveillance and lipid surfactant homeostasis. Alveolar macrophages are surrounded by a coat of hyaluronan that is bound by CD44, a major hyaluronan receptor on immune cells, and this interaction contributes to their survival and the maintenance of normal alveolar macrophage numbers. Alveolar macrophages are conditioned by the alveolar environment to be immunosuppressive, and can phagocytose particulates without alerting an immune response. However, during acute lung infection or injury, an inflammatory immune response is triggered. Hyaluronan levels in the lung are rapidly increased and peak with maximum leukocyte infiltration, suggesting a role for hyaluronan in facilitating leukocyte access to the injury site. Hyaluronan can also be bound by hyaladherins (hyaluronan binding proteins), which create a provisional matrix to facilitate tissue repair. During the subsequent remodeling process hyaluronan concentrations decline and levels return to baseline as homeostasis is restored. In chronic lung diseases, the inflammatory and/or repair phases persist, leading to sustained high levels of hyaluronan, accumulation of associated immune cells and an inability to resolve the inflammatory response.

CD44-mediated hyaluronan binding marks proliferating hematopoietic progenitor cells and promotes bone marrow engraftment.

CD44 is a widely expressed cell adhesion molecule that binds to the extracellular matrix component, hyaluronan. However, this interaction is not constitutive in most immune cells at steady state, as the ability of CD44 to engage hyaluronan is highly regulated. While activated T cells and macrophages gain the ability to bind hyaluronan by CD44, the status in other immune cells is less studied. Here we found a percentage of murine eosinophils, natural killer and natural killer T cells were capable of interacting with hyaluronan at steady state. To further investigate the consequences of hyaluronan binding by CD44 in the hematopoietic system, point mutations of CD44 that either cannot bind hyaluronan (LOF-CD44) or have an increased affinity for hyaluronan (GOF-CD44) were expressed in CD44-deficient bone marrow. Competitive bone marrow reconstitution of irradiated mice revealed an early preference for GOF-CD44 over WT-CD44 expressing cells, and for WT-CD44 over LOF-CD44 expressing cells, in the hematopoietic progenitor cell compartment. The advantage of the hyaluronan-binding cells was observed in the hematopoietic stem and progenitor populations, and was maintained throughout the immune system. Hematopoietic stem cells bound minimal hyaluronan at steady state, and this was increased when the cells were induced to proliferate whereas multipotent progenitors had an increased ability to bind hyaluronan at steady state. In vitro, the addition of hyaluronan promoted their proliferation. Thus, proliferating hematopoietic progenitors bind hyaluronan, and hyaluronan binding cells have a striking competitive advantage in bone marrow engraftment.

Hyaluronan-binding by CD44 reduces the memory potential of activated murine CD8 T cells.

Expansion and death of effector CD8 T cells are regulated to limit immunopathology and cells that escape contraction go on to generate immunological memory. CD44, a receptor for the extracellular matrix component hyaluronan, is a marker of activated and memory T cells. Here, we show with a murine model that the increase in CD44 expression and hyaluronan binding induced upon CD8 T cell activation was proportional to the strength of TCR engagement, thereby identifying the most strongly activated T cells. When CD44-/- and CD44+/+ OT-I CD8 T cells were adoptively transferred into mice challenged with Listeria-OVA, there was a slight increase in the percentage of CD44+/+ cells at the effector site. However, CD44+/+ cells were out-competed by CD44-/- cells after the contraction phase in the lymphoid tissues, and the CD44-/- cells preferentially formed more memory cells. The hyaluronan-binding CD44+/+ CD8 effector T cells showed increased pAkt expression, higher glucose uptake, and were more susceptible to cell death during the contraction phase compared to non-binding CD44+/+ and CD44-/- OT-I CD8 T cells, suggesting that CD44 and its engagement with hyaluronan skews CD8 T cells toward a terminal effector differentiation state that reduces their ability to form memory cells.

The where, when, how, and why of hyaluronan binding by immune cells.

Hyaluronan is made and extruded from cells to form a pericellular or extracellular matrix (ECM) and is present in virtually all tissues in the body. The size and form of hyaluronan present in tissues are indicative of a healthy or inflamed tissue, and the interactions of hyaluronan with immune cells can influence their response. Thus, in order to understand how inflammation is regulated, it is necessary to understand these interactions and their consequences. Although there is a large turnover of hyaluronan in our bodies, the large molecular mass form of hyaluronan predominates in healthy tissues. Upon tissue damage and/or infection, the ECM and hyaluronan are broken down and an inflammatory response ensues. As inflammation is resolved, the ECM is restored, and high molecular mass hyaluronan predominates again. Immune cells encounter hyaluronan in the tissues and lymphoid organs and respond differently to high and low molecular mass forms. Immune cells differ in their ability to bind hyaluronan and this can vary with the cell type and their activation state. For example, peritoneal macrophages do not bind soluble hyaluronan but can be induced to bind after exposure to inflammatory stimuli. Likewise, naïve T cells, which typically express low levels of the hyaluronan receptor, CD44, do not bind hyaluronan until they undergo antigen-stimulated T cell proliferation and upregulate CD44. Despite substantial knowledge of where and when immune cells bind hyaluronan, why immune cells bind hyaluronan remains a major outstanding question. Here, we review what is currently known about the interactions of hyaluronan with immune cells in both healthy and inflamed tissues and discuss how hyaluronan binding by immune cells influences the inflammatory response.