Neutrophilic mice - A model for developing drugs against NETs

Neutrophils represent 50-70% of blood leukocytes and are essential effector cells of the innate immune system. Neutrophils are generated in large numbers in the bone marrow, and are recruited into circulation in baseline numbers²⁵⁵. The release of neutrophils is tightly controlled and only 1 or 2% of all neutrophils in the body are found in the blood under normal homeostatic conditions²⁵⁶. In response to infection or injury, the number of circulating neutrophils rapidly increases through inflammation. As the first line of defense against invading microorganisms, neutrophils control infection by acting as phagocytes, releasing lytic enzymes from their granules, produce reactive oxygen species or generate NETs^257,258. Neutrophils are also key regulators of both acute and chronic inflammation.

By releasing proteases and secreting cytokines such as IL-1ß, IL-18, and TNF-alpha, neutrophils can amplify inflammation^259,260.

Defects in neutrophils quantity and function lead to severe and potentially life-threatening disease in humans, underscoring the important role of the neutrophils in the immune system^{261, 262}. An increase in the absolute number of neutrophils in circulation refers to as neutrophilia. Primary neutrophilia results from abnormalities in the regulation of bone marrow neutrophil production. Secondary or reactive neutrophilia occurs in response to ongoing processes such as acute infections, and chronic inflammation such as rheumatoid arthritis and inflammatory bowel disorders^{261, 263}. NETs are particularly important in the amplification of inflammation. Although NET formation is an essential event in innate immunity, it has adverse effects on the hosts. The release of intracellular components, such as DNA, histones, MPO, and NE, induce injury to the endothelial cells and thrombosis. In the case of persistent NETs in vivo, autoantibodies against NET components are produced^264,265. Hence, impaired regulation of NETs is associated with the pathogenesis of autoimmune diseases such as SLE, vasculitis, RA and gout 222,82, 266. Several studies show that NETs play a role in the pathogenesis of sepsis¹⁶⁰. Indeed, the NET components act as a prognostic marker of sepsi ²⁶⁷.

98 More than a decade of research has uncovered the destructive functions of neutrophils and NETs in inflammatory diseases. However, there has been little progress in explicitly targeting them for therapeutic purpose. There are several experimental challenges in studying NETs, including their short life, isolation of naive neutrophils, stimulus used to activate neutrophils and the detection of NETs in tissue²⁶⁰. For better understanding of the relation of NETosis with human diseases, a simple and quantitative method to evaluate NETs is required. The use of knockout mice helped uncover the role of various proteins and ROS in disease. However, due to the lack of tools to visualize neutrophils it has made it challenging to show that neutrophils are the source of these proteins260,268,269.

An important challenge is the low number of circulating neutrophils in mice. Laboratory mice are the leading model system for biomedical research¹⁸⁹. Many mouse strains are widely used to develop in vivo disease models, to test drugs, and to develop novel therapeutic strategies²⁷⁰. Despite the high conservation in the genome between mice and humans, there exist intrinsic differences in the biology in neutrophils between the two species. Human blood is abundant in neutrophils (50-70% neutrophils) with a mean neutrophil count of 5x10⁹/l in adults, whereas neutrophils in mouse blood are scarce (10-25%)¹⁸⁹. This discrepancy between murine and human neutrophils indicates that findings in mice may not translate to humans. Establishing an optimal mouse model to understand NETs holds the potential to further elucidate the role of NETs in normal and pathologic processes as well as therapeutic targeting.

To study the role of neutrophils in vivo and in vitro, we developed a mouse model with increased neutrophils in circulation by transgenic G-CSF expression. G-CSF is commonly used in clinics to treat neutropenia²¹⁵. It is the key cytokine that stimulates the production and mobilization of neutrophils from the bone marrow²⁷¹,¹⁹⁰. The level of G-CSF is increased during infection, to facilitate for the need for greater number of neutrophils²⁰. G-CSF also activates mature granulocytes. Binding of G-CSF to its receptor prolongs the lifespan of granulocytes and enhances the phagocytic activity and induces degranulation^272,214. Mature neutrophils stay in the bone marrow through the function of chemokine receptors, CXCR2 and CXCR4. Bone marrow stromal cells and osteoblasts produce CXCL12, and that retains the CXCR4-expressing neutrophils in the bone marrow. G-CSF interferes with the CXCR4-CXCL12 interaction and induces

99 neutrophil exit from the bone. Furthermore, when neutrophils need to be mobilized into the blood, the ligands for CXCR2, are expressed by endothelial cells outside the bone marrow. G-CSF induces the upregulation of CXCR2 ligands, reduces the expression of CXCL12 by bone marrow stromal cells and reduced expression of CXCR4 on neutrophils^273,274,24. Therefore, G-CSF treatment is a promising method to overcome the discrepancy between human and mouse neutrophils

Using the pLIVE hepatocyte-specific expression vector, we stably expressed G-CSF as a transgene in the hepatocytes by hydrodynamic tail vein injection. This artificial expression of G-CSF in mice lead to the development of mice with an elevated number of neutrophils in circulation. These mice can mimic humans with regards to neutrophils numbers in circulation. In humans, chronic neutrophilia is characterized by splenomegaly. In line with this, we showed that G-CSF treated mice developed splenomegaly. The spleen weight and size increased with the neutrophil counts in circulation. In addition to this, histological analyses showed the infiltration of neutrophils in vital organs. Spontaneous NET-like structure that stained positively for the DNA specific fluorescent dye Sytox green were present in the neutrophilic mice. This observation suggests that neutrophils in circulation become activated to form NETs, when present at high concentrations. Surprisingly, the neutrophilic mice remained healthy, showed normal behavior, and a normal rate of growth. Measurement of plasma parameters of tissue damage (LDH), liver damage (ALT and AST) as well as renal dysfunction (Creatinine and BUN), indicated no organ damage. In conclusion, we developed a mouse model, exhibiting the characteristic features of chronic neutrophilia in humans, thereby creating a bridge in the immune system between both species. This animal model provides a valuable tool to understand the role of neutrophils and NETs in vivo.

It is well established that NETs in circulation are cytotoxic, yet these mice remain healthy²⁷⁵. Taken together, we hypothesized that the wild-type mice remained healthy despite the formation of NET-like structures in circulation because they are equipped with the machinery to clear NETs in circulation.

100 4.2 DNASE1 and DNASE1L3 provide a therapy to degrade intravascular NETs