The UCSF COMET Consortium, Michael Matthay, David J. E rl e , P re scot t G. Woodruff, Charles Langelier, Kirsten K an ge la ri s, C ar ol yn M.

The coronavirus disease 2019 (COVID-19) pandemic caused by SARS-CoV-2 infections has led to a substantial unmet need for treatments, many of which will require testing in appropriate animal models of this disease. Vaccine trials are already underway, but there remains an urgent need to find other therapeutic approaches to either target SARS-CoV-2 or the complications arising from viral infection, particularly the dysregulated immune response and systemic complications which have been associated with progression to severe COVID-19. At the time of writing, in vivo studies of SARS-CoV-2 infection have been described using macaques, cats, ferrets, hamsters, and transgenic mice expressing human angiotensin I converting enzyme 2 (ACE2). These infection models have already been useful for studies of transmission and immunity, but to date only partly model the mechanisms involved in human severe COVID-19. There is therefore an urgent need for development of animal models for improved evaluation of efficacy of drugs identified as having potential in the treatment of severe COVID-19. These models need to reproduce the key mechanisms of COVID-19 severe acute respiratory distress syndrome and the immunopathology and systemic sequelae associated with this disease. Here, we review the current models of SARS-CoV-2 infection and COVID-19-related disease mechanisms and suggest ways in which animal models can be adapted to increase their usefulness in research into COVID-19 pathogenesis and for assessing potential treatments.

While platelets are the cellular mediators of thrombosis, platelets are also immune cells. Platelets interact both directly and indirectly with immune cells, impacting their activation and differentiation, as well as all phases of the immune response. Megakaryocytes (Mks) are the cell source of circulating platelets, and until recently Mks were typically only considered as bone marrow (BM) resident cells. However, platelet producing Mks also reside in the lung, and lung Mks express greater levels of immune molecules compared to BM Mks. We therefore sought to define the immune functions of lung Mks. Using single cell RNA-Seq of BM and lung myeloid enriched cells, we found that lung Mks (MkL) had gene expression patterns that are similar to antigen presenting cells (APC). This was confirmed using imaging and conventional flow cytometry. The immune phenotype of Mks was plastic and driven by the tissue immune environment as evidenced by BM Mks having a MkL like phenotype under the influence of pathogen receptor challenge and lung associated immune molecules, such as IL-33. Our in vitro and in vivo assays demonstrated that MkL internalized and processed both antigenic proteins and bacterial pathogens. Furthermore, MkL induced CD4 + T cell activation in a MHC II dependent manner both in vitro and in vivo. These data indicated that Mks in the lung had key immune regulatory roles dictated in part by the tissue environment.

Key Points• P-Rex and Vav Rac-GEFs cooperate in leukocyte recruitment during inflammation by facilitating leukocyte adhesion to the vascular endothelium.• P-Rex/Vav expression in platelets is required for vascular adhesion and recruitment of neutrophils and eosinophils into lung tissue.The small GTPase Rac is required for neutrophil recruitment during inflammation, but its guanine-nucleotide exchange factor (GEF) activators seem dispensable for this process, which led us to investigate the possibility of cooperation between Rac-GEF families. Thioglycollate-induced neutrophil recruitment into the peritoneum was more severely impaired in P-Rex1 2/2 Vav1 2/2 (P1V1) or P-Rex1 2/2 Vav3 2/2 (P1V3) mice than in P-Rex null or Vav null mice, suggesting cooperation between P-Rex and Vav Rac-GEFs in this process. Neutrophil transmigration and airway infiltration were all but lost in P1V1 and P1V3 mice during lipopolysaccharide (LPS)-induced pulmonary inflammation, with altered intercellular adhesion molecule 1-dependent slow neutrophil rolling and strongly reduced L-and E-selectin-dependent adhesion in airway postcapillary venules. Analysis of adhesion molecule expression, neutrophil adhesion, spreading, and migration suggested that these defects were only partially neutrophil-intrinsic and were not obviously involving vascular endothelial cells. Instead, P1V1 and P1V3 platelets recapitulated the impairment of LPS-induced intravascular neutrophil adhesion and recruitment, showing P-Rex and Vav expression in platelets to be crucial. Similarly, during ovalbumin-induced allergic inflammation, pulmonary recruitment of P1V1 and P1V3 eosinophils, monocytes, and lymphocytes was compromised in a plateletdependent manner, and airway inflammation was essentially abolished, resulting in improved airway responsiveness. Therefore, platelet P-Rex and Vav family Rac-GEFs play important proinflammatory roles in leukocyte recruitment. (Blood. 2015;125(7):1146-1158) IntroductionDuring inflammation, neutrophils are rapidly recruited from the bloodstream into inflamed tissues where they mount proinflammatory and antimicrobial responses.1 Recruitment occurs in a cascade of steps, beginning with the upregulation of P-selectin on the surface of endothelial cells that line postcapillary venules. P-selectin captures neutrophils from the bloodstream by engaging P-selectin glycoprotein ligand 1 (PSGL1) on their surface, enabling them to roll along the intraluminal wall. When captured, L-selectin on the neutrophil surface engages endothelial PSGL1 to support rolling, and endothelial E-selectin engages neutrophil PSGL1, among other counterligands, to slow rolling down. Binding of the neutrophil integrins LFA1 and Mac1 to their endothelial ligand intercellular adhesion molecule 1 (ICAM1) confers firm adhesion, and Mac1 enables the cells to crawl along the vessel wall before they actively transmigrate into the inflamed tissue by para-or transcellular routes.2 This recruitment cascade has largely been elucidated in the inflamed cremaster muscle and mesen...

Platelet activation occurs during host defence and in various inflammatory disorders. In animal models of infection and inflammation, experimental depletion of platelets leads to significantly reduced leukocyte recruitment and impaired clearance of pathogens from the lung. It is now appreciated that purinergic receptor activation is required for leukocyte activation, motility and adhesion, and platelet interactions with leukocytes can be modulated by purinergic stimulation of platelets. Here, we have investigated the role of platelet P2Y P2Y, P2Y, and P2X receptors on leukocyte recruitment and chemotaxis. Mice were administered either vehicle controls or selective P2Y P2Y, P2Y, or P2X antagonists intravenously before intranasal administration of lipopolysaccharide (LPS) to investigate the effect of these drugs on pulmonary leukocyte recruitment, peripheral platelet counts, bleeding times, and ex vivo platelet aggregation. Separately, platelets were incubated with P2Y P2Y, P2X antagonists, or P2Y agonists to assess effects on platelet-induced neutrophil chemotaxis in vitro. Pulmonary neutrophil recruitment induced by intranasal LPS administration was inhibited in mice administered either with P2Y or P2Y antagonists, but not with P2Y or P2X antagonists. Furthermore, the administration of either a P2Y or a P2Y antagonist reversed the incidence of peripheral thrombocytopaenia associated with LPS exposure. Bleeding times were significantly increased in mice administered P2Y P2Y, or P2X antagonists, whilst ex vivo platelet aggregation to ADP was significantly reduced. These haemostatic responses remained unaltered following antagonism of P2Y. In vitro chemotaxis assays revealed direct antagonism of platelet P2Y but not P2Y or P2X receptors suppressed platelet-dependent neutrophil motility towards Macrophage derived chemokine (MDC, CCL22). Furthermore, the stimulation of platelets with selective P2Y agonists (UDP-glucose, MRS2690) resulted in significant platelet-dependent neutrophil chemotaxis. These results reveal a role for P2Y and P2Y activation of platelets following exposure to LPS, whilst haemostatic indices were unaffected by inhibition of platelet function with the P2Y antagonist in response to LPS.

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Platelets have been implicated in pulmonary inflammatory cell recruitment after exposure to allergic and nonallergic stimuli, but little is known about the role of platelets in response to pulmonary infection with Pseudomonas aeruginosa. In this study, we have investigated the impact of the experimental depletion of circulating platelets on a range of inflammatory and bacterial parameters, and their subsequent impact on mortality in a murine model of pulmonary infection with P. aeruginosa. P. aeruginosa infection in mice induced a mild, but significant, state of peripheral thrombocytopenia in addition to pulmonary platelet accumulation. Increased platelet activation was detected in infected mice through increased levels of the platelet-derived mediators, platelet factor-4 and β-thromboglobulin, in BAL fluid and blood plasma. In mice depleted of circulating platelets, pulmonary neutrophil recruitment was significantly reduced 24 hours after infection, whereas the incidence of systemic dissemination of bacteria was significantly increased compared with non-platelet-depleted control mice. Furthermore, mortality rates were increased in bacterial-infected mice depleted of circulating platelets. This work demonstrates a role for platelets in the host response toward a gram-negative bacterial respiratory infection.

Platelets are recruited to inflammatory foci and contribute to host defence and inflammatory responses. Compared to platelet recruitment in haemostasis and thrombosis, the mechanisms of platelet recruitment in inflammation and host defence are poorly understood. Neutrophil recruitment to lung airspaces following inhalation of bacterial LPS requires platelets and PSGL-1 in mice. Given this association between platelets and neutrophils, we investigated whether recruitment of platelets to lungs of mice following LPS inhalation was dependent on PSGL-1, P-selectin, or interaction with neutrophils.BALB/c mice were administered intranasal LPS (O55:B5, 5 mg/kg) and 48 hours later lungs were collected, and platelets and neutrophils were quantified in tissue sections by immunohistochemistry.The effects of functional blocking antibody treatments targeting the platelet-neutrophil adhesion molecules P-selectin, or PSGL-1, or treatment with a neutrophil depleting antibody targeting Ly6G, were tested on the extent of LPS-induced lung platelet recruitment. Separately in Pf4-Cre×mTmG mice, two-photon intravital microscopy was used to image platelet adhesion in live lungs.Inhalation of LPS caused both platelet and neutrophil recruitment to the lung vasculature. However, decreasing lung neutrophil recruitment by blocking PSGL-1, P-selectin, or depleting blood neutrophils had no effect on lung platelet recruitment. Lung intravital imaging revealed increased adhesion of platelets in the lung microvasculature which was not associated with thrombus formation.In conclusion, platelet recruitment to lungs in response to LPS occurs through mechanisms distinct from those mediating neutrophil recruitment, or the occurrence of pulmonary emboli.