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

Regulation of the NLRP3 inflammasome and IL-1β production and release during Toxoplasma gondii infection of human monocytes

Toxoplasma gondii is an obligate intracellular eukaryotic parasite that is estimated to infect one-third of the global population and is especially life-threatening in developing fetuses and immunocompromised individuals. Innate immune cells contribute to host defense against T. gondii infection. Specifically, monocytes are rapidly recruited to sites of infection and CCR2 or CCL2 KO mice are more susceptible to T. gondii infection. Monocytes protect against infection by initiating a robust inflammatory response which is in part mediated by release of IL-1β during activation of the NLRP3 inflammasome. IL-1β is also implicated in the development of many autoimmune disorders like rheumatoid arthritis and atherosclerosis, but there is still much that is unknown about how human monocytes produce, process and release IL-1β. We have identified that T. gondii induces IL-1β production 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 transcription and production of pro-IL-1β protein. Inhibition of PKCδ, CARD9/MALT-1 and IKK also reduced p65 phosphorylation and pro-IL-1β production in T. gondii-infected primary monocytes, and genetic knock-out (KO) 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. We have also found that primary human monocytes treated with a caspase-8 inhibitor released less IL-1β after T. gondii infection than control cells. Similarly, caspase-1 and caspase-8 KO human monocytic THP-1 cells, but not caspase-4 or -5 KO cells were impaired in their release of IL-1β after infection compared to empty vector (EV) THP-1 cells. We found caspase-8 deficiency did not significantly affect the pro-IL-1β transcripts or production of pro-IL-1β protein. Similarly, caspase-8 had no significant affect on NLRP3 inflammasome activation or cleavage of pro-IL-1β to mature IL-1β during T. gondii infection. Instead, caspase-8 deficiency appeared to stunt the release mechanism of IL-1β from infected cells as mature-IL-1β would accumulate intracellularly in these KO cells. IL-1β release from T. gondii-infected primary human monocytes did require an NLRP3-ASC-caspase-1 inflammasome. While the release mechanism of IL-1β during NLRP3 inflammasome activation often requires cleavage of gasdermin D (GSDMD), formation of pores in the cell membrane and induction of an inflammatory form of cell death known as pyroptosis, human monocytes released IL-1β independent of these factors. 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 also describes a novel role for caspase-8 in IL-1β release from T. gondii-infected monocytes and contributes to the growing notion that IL-1β can be released from human myeloid cells independent of cell death. Collectively, this research expands our understanding of the molecular basis for human innate immune regulation of inflammation and host defense during parasite infection

Striatal Neurodevelopment Is Dysregulated in Purine Metabolism Deficiency and Impacts DARPP-32, BDNF/TrkB Expression and Signaling: New Insights on the Molecular and Cellular Basis of Lesch-Nyhan Syndrome

<div><p>Lesch-Nyhan Syndrome (LNS) is a neurodevelopmental disorder caused by mutations in the gene encoding the purine metabolic enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT). This syndrome is characterized by an array of severe neurological impairments that in part originate from striatal dysfunctions. However, the molecular and cellular mechanisms underlying these dysfunctions remain largely unidentified. In this report, we demonstrate that HPRT-deficiency causes dysregulated expression of key genes essential for striatal patterning, most notably the striatally-enriched transcription factor B-cell leukemia 11b (Bcl11b). The data also reveal that the down-regulated expression of Bcl11b in HPRT-deficient immortalized mouse striatal (STH<i>dh</i>) neural stem cells is accompanied by aberrant expression of some of its transcriptional partners and other striatally-enriched genes, including the gene encoding dopamine- and cAMP-regulated phosphoprotein 32, (DARPP-32). Furthermore, we demonstrate that components of the BDNF/TrkB signaling, a known activator of DARPP-32 striatal expression and effector of Bcl11b transcriptional activation are markedly increased in HPRT-deficient cells and in the striatum of HPRT knockout mouse. Consequently, the HPRT-deficient cells display superior protection against reactive oxygen species (ROS)-mediated cell death upon exposure to hydrogen peroxide. These findings suggest that the purine metabolic defect caused by HPRT-deficiency, while it may provide neuroprotection to striatal neurons, affects key genes and signaling pathways that may underlie the neuropathogenesis of LNS.</p></div

Gene & Protein expression for HPRT and ASCL1.

<p>(<b>A</b>) Gene expression for HPRT and ASCL1 in control (CTL, open bars) and HPRT knockdown (HPRTKD, closed bars) striatal STHdh cells. Bars represent mean ±SEM of duplicate PCR measurements carried out independently twice (n = 4). mRNA expression is normalized to GAPDH level (*P<0.05, t-test). (<b>B</b>) Immuno-blot and quantification of HPRT and ASCL1 protein expression in striatal STHdh cells. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>C</b>) Gene expression for DLX2, FOXG1 and GSX1 in control (CTL, open bars) and HPRT knockdown (HPRTKD, closed bars) Striatal cells. Bars represent mean ±SEM (n = 4) *P<0.05, t-test.</p

Restoration of Bcl11b expression in HPRT-deficient striatal cells:

<p>(<b>A</b>) Immuno-blot and quantification of Bcl11b expression in control (CTL) and HPRT-deficient striatal cells transfected with plasmid encoding Bcl11b (Bcl11b) and GFP (GFP). The quantification bar graphs are shown means± SEM (n = 3, *P<0.05, ANOVA, Tukey post-hoc test). (<b>B</b>) Immuno-blot and quantification of Y705/706 TrkB after H₂O₂ exposure for control (CTL), HPRT-deficient cells transfected with Bcl11b (Bcl11b) and GFP (GFP). The quantification bar graphs are shown as means ± SEM (n = 4, *P<0.05, ANOVA, Tukey post-hoc test). (<b>C</b>) quantification of the number of Sytox green fluorescent cells upon treatment of control (CTL) and HPRT-deficient cells transfected with Bcl11b (BCL11b) and GFP (GFP) plasmid. Data show that the rescue of Bcl11b expression in HPRT-deficient cells does not lead to added protection from cell death triggered by H₂O₂ (ns =  non significant, *p<0.05, ANOVA). (<b>D</b>) Gene expression of DARPP-32 in control (CTL), GFP and Bcl11b (rescued) transfected cells. Data show that the rescued expression of Bcl11b in HPRT-deficient cells leads to restoration of DARPP-32 expression in level similar to control, the data are shown as means ± SEM ((ns =  non significant, n = 4, *P<0.05, ANOVA, Tukey post-hoc test). (<b>E</b>) Gene expression for Actn2, Arpp19, Foxp1, Ngef, Pde10a, and Rgs9 in control (CTL, open bars), GFP (GFP, closed bars) and Bcl11b (Bcl11b, grey bars) transfected striatal cells. Bars represent mean ±SEM (n = 4). *P<0.05, ANOVA.</p

Gene and Protein Expression for <i>Ppp1r1b</i>/DARPP-32.

<p>(<b>A</b>) Gene expression for <i>Ppp1r1b</i>/DARPP-32 in control striatal STHdh cells (CTL, open bars) and HPRT-knockdown (HPRTKD, closed bars). Bars represent mean ±SEM (n = 4). mRNA expression is normalized to GAPDH mRNA level (*P<0.05, t-test). (<b>B</b>) Immuno-blot and quantification of DARPP-32 protein expression in striatal cells. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>C</b>) Gene expression for <i>Ppp1r1b</i>/DARPP-32 in the striatum of wild-type (WT, open bars) and HPRT knockout (HPRTKO, closed bars) mice. Bars represent mean ±SEM (n = 4). mRNA expression is normalized to GAPDH RNA level (*P<0.05, t-test). (<b>D</b>) Immuno-blot and quantification of DARPP-32 protein expression in the striatum of WT and HPRTKO mice. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>E</b>) DARPP-32 phosphoprotein (Thr34) expression in response to D1R (SKF393298) agonist treatment (50 µM). Immuno-blot and quantification of Thr34-DARPP-32 in control and HPRT-deficient striatal cells, the quantification bar graphs are shown as means ± SEM (n = 6, *P<0.05). (<b>F</b>) Thr34-DARPP-32 phosphoprotein expression in striatal tissue of WT (open bar) and HPRTKO (closed bar) mice (n = 3, *P<0.05).</p

Schematic and summary model of HPRT-deficiency role in dysregulating striatal gene expression and signaling.

<p>HPRT-deficiency alters the purine pool; and via mechanisms that are still ill-defined, these alterations affect the gene expression of keys striatal-enriched transcription factors such as Ascl1, Bcl11b and Foxp1, notably during neurodevelopment. Later in mature neurons, these transcription factors control (directly or indirectly) the expression of several genes among them, <i>Darpp-32</i> and <i>Bdnf</i>, whose the dysregulation affects striatal neurons signaling and neurotransmission, thus contributing to LNS neurological phenotype.</p

Effects of hydrogen peroxide-mediated cellular toxicity in HPRT-deficient striatal cells:

<p>(<b>A</b>) (Y705/706) TrkB phosphoprotein expression in response to hydrogen peroxide H₂O₂ treatment (100 µM). Immuno-blot and quantification of Y705/706 TrkB phosphoprotein in control and HPRT-deficient striatal STHdh cells. The quantification bar graphs are shown as means ± SEM (n = 6, *P<0.05). (<b>B</b>) Enhanced neuroprotective effects against reactive oxygen species (ROS)-mediated cell death in HPRT-deficient striatal cells. Control (CTL) and HPRT-knock-down cells (HPRTKD) were treated with hydrogen peroxide H₂O₂ for 4 hours and then exposed to Sytox green for 10 min. Figure shows microscopy images of DAPI staining and green fluorescence which is a measure of the overall cell death level. There is reduced green fluorescence in HPRT-deficient cells relative to control after stimulation with H₂O₂. (Bar scale, 100 µm). This is confirmed by the quantification of the number of Sytox green fluorescent cells as illustrated in the appended graph. Error bars represent mean ± SEM of duplicate measurements of two independent experiments (n = 4). The asterisks (*) represent statistical significance between H₂O₂ treated cells (p<0.05, t-test).</p

Gene and Protein Expression for BDNF and TRKB.

<p>(<b>A</b>) Gene expression for <i>Bdnf</i> and <i>Trkb</i> in control (CTL, open bars) and HPRT knockdown (HPRTKD, closed bars) striatal STHdh cells. Bars represent mean ±SEM (n = 4). mRNA expression is normalized to GAPDH RNA level (*P<0.05, t-test). (<b>B</b>) Immuno-blot and quantification of BDNF and TRKB protein expression in striatal cells. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>C</b>) Gene expression for <i>Bdnf</i> and <i>Trkb</i> in the striatum of wild-type (WT, open bars) and HPRT knockout (HPRTKO, closed bars) mice. Bars represent mean ±SEM of duplicate PCR measurements (n = 4). mRNA expression is normalized to GAPDH mRNA level (*P<0.05, t-test). (<b>D</b>) Immuno-blot and quantification of BDNF and TrkB protein expression in the striatum of WT and HPRTKO mice. The quantification bar graphs are shown as means ± SEM (n = 3, *P<0.05, t-test). (<b>E</b>) (Y705/706) TrkB phosphoprotein expression in response to BDNF treatment (10 ng/ml). Immuno-blot and quantification of Y705/706) TrkB phosphoprotein in control and HPRT-deficient striatal cells, the quantification bar graphs are shown as means ± SEM (n = 6, *P<0.05).</p