Single-cell immune profiling reveals long-term changes in myeloid cells and identifies a novel subset of CD9(+) monocytes associated with COVID-19 hospitalization

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can result in severe immune dysfunction, hospitalization, and death. Many patients also develop long-COVID-19, experiencing symptoms months after infection. Although significant progress has been made in understanding the immune response to acute SARS-CoV-2 infection, gaps remain in our knowledge of how innate immunity influences disease kinetics and severity. We hypothesized that cytometry by time-of-flight analysis of PBMCs from healthy and infected subjects would identify novel cell surface markers and innate immune cell subsets associated with COVID-19 severity. In this pursuit, we identified monocyte and dendritic cell subsets that changed in frequency during acute SARS-CoV-2 infection and correlated with clinical parameters of disease severity. Subsets of nonclassical monocytes decreased in frequency in hospitalized subjects, yet increased in the most severe patients and positively correlated with clinical values associated with worse disease severity. CD9, CD163, PDL1, and PDL2 expression significantly increased in hospitalized subjects, and CD9 and 6-Sulfo LacNac emerged as the markers that best distinguished monocyte subsets amongst all subjects. CD9+ monocytes remained elevated, whereas nonclassical monocytes remained decreased, in the blood of hospitalized subjects at 3-4 months postinfection. Finally, we found that CD9+ monocytes functionally released more IL-8 and MCP-1 after LPS stimulation. This study identifies new monocyte subsets present in the blood of COVID-19 patients that correlate with disease severity, and links CD9+ monocytes to COVID-19 progression

Toxoplasma gondii activates a Syk-CARD9-NF-κB signaling axis and gasdermin D-independent release of IL-1β during infection of primary human monocytes.

IL-1β is a potent pro-inflammatory cytokine that promotes immunity and host defense, and its dysregulation is associated with immune pathology. Toxoplasma gondii infection of myeloid cells triggers the production and release of IL-1β; however, the mechanisms regulating this pathway, particularly in human immune cells, are incompletely understood. We have identified a novel pathway of T. gondii induction of IL-1β via a Syk-CARD9-NF-κB signaling axis in primary human peripheral blood monocytes. Syk was rapidly phosphorylated during T. gondii infection of primary monocytes, and inhibiting Syk with the pharmacological inhibitors R406 or entospletinib, or genetic ablation of Syk in THP-1 cells, reduced IL-1β release. Inhibition of Syk in primary cells or deletion of Syk in THP-1 cells decreased parasite-induced IL-1β transcripts and the production of pro-IL-1β. Furthermore, inhibition of PKCδ, CARD9/MALT-1 and IKK reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knockout of PKCδ or CARD9 in THP-1 cells also reduced pro-IL-1β protein levels and IL-1β release during T. gondii infection, indicating that Syk functions upstream of this NF-κB-dependent signaling pathway for IL-1β transcriptional activation. IL-1β release from T. gondii-infected primary human monocytes required the NLRP3-caspase-1 inflammasome, but interestingly, was independent of gasdermin D (GSDMD) cleavage and pyroptosis. Moreover, GSDMD knockout THP-1 cells released comparable amounts of IL-1β to wild-type THP-1 cells after T. gondii infection. Taken together, our data indicate that T. gondii induces a Syk-CARD9/MALT-1-NF-κB signaling pathway and activation of the NLRP3 inflammasome for the release of IL-1β in a cell death- and GSDMD-independent manner. This research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection

mTORC1 Inhibition Induces Resistance to Methotrexate and 6-Mercaptopurine in Ph+ and Ph-like B-ALL.

Elevated activity of mTOR is associated with poor prognosis and higher incidence of relapse in B-cell acute lymphoblastic leukemia (B-ALL). Thus, ongoing clinical trials are testing mTOR inhibitors in combination with chemotherapy in B-ALL. However, the combination of mTOR inhibitors with standard of care chemotherapy drugs has not been studied extensively in high-risk B-ALL subtypes. Therefore, we tested whether mTOR inhibition can augment the efficacy of current chemotherapy agents in Ph+ and Ph-like B-ALL models. Surprisingly, inhibiting mTOR complex 1 (mTORC1) protected B-ALL cells from killing by methotrexate and 6-mercaptopurine, two antimetabolite drugs used in maintenance chemotherapy. The cytoprotective effects correlated with decreased cell-cycle progression and were recapitulated using cell-cycle inhibitors, palbociclib or aphidicolin. Dasatinib, a tyrosine kinase inhibitor currently used in Ph+ patients, inhibits ABL kinase upstream of mTOR. Dasatinib resistance is mainly caused by ABL kinase mutations, but is also observed in a subset of ABL unmutated cases. We identified dasatinib-resistant Ph+ cell lines and patient samples in which dasatinib can effectively reduce ABL kinase activity and mTORC1 signaling without causing cell death. In these cases, dasatinib protected leukemia cells from killing by 6-mercaptopurine. Using xenograft models, we observed that mTOR inhibition or dasatinib increased the numbers of leukemia cells that emerge after cessation of chemotherapy treatment. These results demonstrate that inhibitors targeting mTOR or upstream signaling nodes should be used with caution when combined with chemotherapeutic agents that rely on cell-cycle progression to kill B-ALL cells. Mol Cancer Ther; 16(9); 1942-53. ©2017 AACR

mTORC1 Inhibition Induces Resistance to Methotrexate and 6-Mercaptopurine in Ph+ and Ph-like B-ALL.

Elevated activity of mTOR is associated with poor prognosis and higher incidence of relapse in B-cell acute lymphoblastic leukemia (B-ALL). Thus, ongoing clinical trials are testing mTOR inhibitors in combination with chemotherapy in B-ALL. However, the combination of mTOR inhibitors with standard of care chemotherapy drugs has not been studied extensively in high-risk B-ALL subtypes. Therefore, we tested whether mTOR inhibition can augment the efficacy of current chemotherapy agents in Ph+ and Ph-like B-ALL models. Surprisingly, inhibiting mTOR complex 1 (mTORC1) protected B-ALL cells from killing by methotrexate and 6-mercaptopurine, two antimetabolite drugs used in maintenance chemotherapy. The cytoprotective effects correlated with decreased cell-cycle progression and were recapitulated using cell-cycle inhibitors, palbociclib or aphidicolin. Dasatinib, a tyrosine kinase inhibitor currently used in Ph+ patients, inhibits ABL kinase upstream of mTOR. Dasatinib resistance is mainly caused by ABL kinase mutations, but is also observed in a subset of ABL unmutated cases. We identified dasatinib-resistant Ph+ cell lines and patient samples in which dasatinib can effectively reduce ABL kinase activity and mTORC1 signaling without causing cell death. In these cases, dasatinib protected leukemia cells from killing by 6-mercaptopurine. Using xenograft models, we observed that mTOR inhibition or dasatinib increased the numbers of leukemia cells that emerge after cessation of chemotherapy treatment. These results demonstrate that inhibitors targeting mTOR or upstream signaling nodes should be used with caution when combined with chemotherapeutic agents that rely on cell-cycle progression to kill B-ALL cells. Mol Cancer Ther; 16(9); 1942-53. ©2017 AACR