Cystatin C is glucocorticoid-responsive, directs recruitment of Trem2+ macrophages and predicts failure of cancer immunotherapy (preprint)

Summary Cystatin C (CyC) is a secreted cysteine protease inhibitor and its biological functions remain insufficiently characterized. Plasma CyC is elevated in many patients, especially when receiving glucocorticoid (GC) treatment. Endogenous GCs are essential for life and are appropriately upregulated in response to systemic stress. Here we empirically connect GCs with systemic regulation of CyC. We used genome-wide association and structural equation modeling to determine the genetics of the latent trait CyC production in UK Biobank. CyC production and a polygenic score (PGS) capturing germline predisposition to CyC production predicted elevated all-cause and cancer-specific mortality. We then demonstrated that CyC is a direct target of GC receptor, with GC-responsive CyC secretion exhibited by macrophages and cancer cells. Using isogenic CyC-knockout tumors, we discovered a markedly attenuated tumor growth in vivo and found abrogated recruitment of Trem2+ macrophages, which have been previously linked to failure of cancer immunotherapy. Finally, we showed that the CyC-production PGS predicted checkpoint immunotherapy failure in a combined clinical trial cohort of 685 metastatic cancer patients. Taken together, our results demonstrate that CyC may be a direct effector of GC-induced immunosuppression, acting through recruitment of Trem2+ macrophages, and therefore could be a target for combination cancer immunotherapy.

A functionally distinct neutrophil landscape in severe COVID-19 reveals opportunities for adjunctive therapies (preprint)

ABSTRACT Acute respiratory distress syndrome (ARDS) is a life-threatening syndrome of respiratory failure and diffuse alveolar damage that results from dysregulated local and systemic immune activation, causing pulmonary vascular, parenchymal and alveolar damage. SARS-CoV-2 infection has become the dominant cause of ARDS worldwide, and emerging evidence implicates neutrophils and their cytotoxic arsenal of effector functions as central drivers of immune-mediated lung injury in COVID-19 ARDS. However, a key outstanding question is whether COVID-19 drives a unique program of neutrophil activation or effector functions that contributes to the severe pathogenesis of this pandemic illness, and whether this unique neutrophil response can be targeted to attenuate disease. Using a combination of high-dimensional single cell analysis and ex vivo functional assays of neutrophils from patients with COVID-19 ARDS compared to non-COVID ARDS (caused by bacterial pneumonia), we identified a functionally distinct landscape of neutrophil activation in COVID-19 ARDS that was intrinsically programmed during SARS-CoV-2 infection. Furthermore, neutrophils in COVID-19 ARDS were functionally primed to produce high amounts of neutrophil extracellular traps (NETs). Surprisingly, this unique pathological program of neutrophil priming escaped conventional therapy with dexamethasone, thereby revealing a promising target for adjunctive immunotherapy in severe COVID-19.

An Immune Cell Atlas Reveals Dynamic COVID-19 Specific Neutrophil Programming Amendable to Dexamethasone Therapy (preprint)

SARS-CoV-2 is a novel coronavirus that causes acute respiratory distress syndrome (ARDS), death and long-term sequelae. Innate immune cells are critical for host defense but are also the primary drivers of ARDS. The relationships between innate cellular responses in ARDS resulting from COVID-19 compared to other causes of ARDS, such as bacterial sepsis is unclear. Moreover, the beneficial effects of dexamethasone therapy during severe COVID-19 remain speculative but understanding the mechanistic effects could improve evidence-based therapeutic interventions. To interrogate these relationships, we developed an scRNAseq atlas that is freely accessible (biernaskielab.ca/COVID_neutrophil). We discovered that compared to bacterial ARDS, COVID-19 was associated with distinct neutrophil polarization characterized by either interferon (IFN) or prostaglandin (PG) active states. Neutrophils from bacterial ARDS had higher expression of antibacterial molecules such as PLAC8 and CD83. Dexamethasone therapy in COVID patients rapidly altered the IFNactive state, downregulated interferon responsive genes, and activated the IL1R2+ve neutrophils. Dexamethasone also induced the emergence of immature neutrophils expressing immunosuppressive molecules ARG1 and ANXA1, which were not present in healthy controls. Moreover, dexamethasone remodeled global cellular interactions by changing neutrophils from information receivers into information providers. Importantly, male patients had higher proportions of IFN-active neutrophils and a greater degree of steroid-induced immature neutrophil expansion. Indeed, the highest proportion of IFN-active neutrophils was associated with mortality. These results define neutrophil states unique to COVID-19 when contextualized to other life-threatening infections, thereby enhancing the relevance of our findings at the bedside. Furthermore, the molecular benefits of dexamethasone therapy are also defined. The identified molecular pathways can now be targeted to develop improved therapeutics.

SARS-CoV-2 Infection of Circulating Immune Cells is Not Responsible for Virus Dissemination in Severe COVID-19 Patients (preprint)

In late 2019 a novel coronavirus (SARS-CoV-2) emerged, and has since caused a global pandemic. Understanding the pathogenesis of COVID-19 disease is necessary to inform development of therapeutics, and management of infected patients. Using scRNAseq of blood drawn from SARS-CoV-2 patients, we asked whether SARS-CoV-2 may exploit immune cells as a ‘Trojan Horse’ to disseminate and access multiple organ systems. Our data suggests that circulating cells are not actively infected with SARS-CoV-2, and do not appear to be a source of viral dissemination.Funding: This work was generously funded by the Calgary Firefighter’s Burn Treatment Society and the Thistledown Foundation.Conflict of Interest: The authors declare that they have no competing interests.Ethical Approval: All experiments involving or human samples received approval from the Conjoint Health Ethics Review Board at the University of Calgary.

SARS-CoV-2 infection of circulating immune cells is not responsible for virus dissemination in severe COVID-19 patients (preprint)

In late 2019 a novel coronavirus (SARS-CoV-2) emerged, and has since caused a global pandemic. Understanding the pathogenesis of COVID-19 disease is necessary to inform development of therapeutics, and management of infected patients. Using scRNAseq of blood drawn from SARS-CoV-2 patients, we asked whether SARS-CoV-2 may exploit immune cells as a 'Trojan Horse' to disseminate and access multiple organ systems. Our data suggests that circulating cells are not actively infected with SARS-CoV-2, and do not appear to be a source of viral dissemination.