Neutrophils are the first wave of recruited immune cells to sites of injury or infection and are crucial players in controlling bacterial and fungal infections. Although the role of neutrophils during bacterial or fungal infections is well understood, their impact on antiviral immunity is much less studied. Furthermore, neutrophil function in tumor pathogenesis and cancer treatment has recently received much attention, particularly within the context of oncolytic virus infection where neutrophils produce antitumor cytokines and enhance oncolysis. In this review, multiple functions of neutrophils in viral infections and immunity are discussed. Understanding the role of neutrophils during viral infection may provide insight into the pathogenesis of virus infections and the outcome of virus-based therapies.

The influenza A virus (IAV) causes a respiratory tract infection with approximately 10% of the population infected by the virus each year. Severe IAV infection is characterized by excessive inflammation and tissue pathology in the lungs. Platelet and neutrophil recruitment to the lung are involved in the pathogenesis of IAV, but the specific mechanisms involved have not been clarified. Using confocal intravital microscopy in a mouse model of IAV infection, we observed profound neutrophil recruitment, platelet aggregation, neutrophil extracellular trap (NET) production and thrombin activation within the lung microvasculature in vivo. Importantly, deficiency or antagonism of the protease-activated receptor 4 (PAR4) reduced platelet aggregation, NET production, and neutrophil recruitment. Critically, inhibition of thrombin or PAR4 protected mice from virus-induced lung tissue damage and edema. Together, these data imply thrombin-stimulated platelets play a critical role in the activation/recruitment of neutrophils, NET release and directly contribute to IAV pathogenesis in the lung.

Non-alcoholic fatty liver disease is a spectrum of liver pathology ranging from simple steatosis to steatohepatitis and can progress to diseases associated with poor outcomes including cirrhosis and hepatocellular carcinoma (HCC). NAFLD research has typically focused on the pathophysiology associated with lipid metabolism, using traditional measures such as histology and serum transaminase assessment; these methods have provided key information regarding NAFLD progression. Although valuable, these techniques are limited in providing further insight into the mechanistic details of inflammation associated with NAFLD. Intravital microscopy (IVM) is an advanced tool that allows for real-time visualization of cellular behavior and interaction in a living animal. Extensive IVM imaging has been conducted in liver, but, in the context of NAFLD, this technique has been regularly avoided due to significant tissue autofluorescence, a phenomenon that is exacerbated with steatosis. Here, we demonstrate that, using multiple imaging platforms and optimization techniques to minimize autofluorescence, IVM in fatty liver is possible. Successful fatty liver intravital imaging provides details on cell trafficking, recruitment, function, and behavior in addition to information about blood flow and vessel dynamics, information which was previously difficult to obtain. As more than 30% of the global population is overweight/obese, there is a significant proportion of the population at risk for NAFLD and complications due to NAFLD (liver decompensation, cirrhosis, HCC). IVM has the potential to elucidate the poorly understood mechanisms surrounding liver inflammation and NAFLD progression and possesses the potential to identify key processes that may be targeted for future therapeutic interventions in NAFLD patients.

Recent advances in imaging technology have made it possible to track cellular recruitment and behavior within the vasculature of living animals in real-time. Using approaches such as resonant scanning confocal and multiphoton intravital microscopy (IVM), we are now able to observe cells within the intact tumor microenvironment of a mouse. We are able to follow these cells for extended periods of time (hours) and can characterize how specific cell types (T cells, neutrophils, monocytes) interact with the tumor vasculature and cancer cells. This approach provides greater insight into specific cellular behaviors and cell–cell interactions than conventional techniques such as histology and flow cytometry. In this report, we describe the surgical preparation of animals to expose the tumor and both resonant scanning confocal and multiphoton imaging approaches used to track leukocyte recruitment, adhesion, and behavior within the tumor microenvironment. We present techniques for the measurement and quantification of leukocyte behavior within the bloodstream and tumor interstitium. The use of IVM to study leukocyte behavior within the tumor microenvironment provides key information not attainable with other approaches, that will help shape the development of better, more effective anticancer drugs and therapeutic approaches.

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