Citations

OBJECTIVES: Critically ill patients with COVID-19 experience high rates of delirium and coma. Whether delirium occurs through novel mechanisms in COVID-19 is not known. We analyzed the relationship among biomarkers of inflammation (C-reactive protein [CRP]), hypercoagulability (d-dimer), and lung macrophage activation (ferritin), and the primary composite outcome of delirium/coma next day. We also measured associations between biomarkers and next day delirium and coma independently, and delirium severity. DESIGN: Retrospective, observational cohort study. SETTING: ICUs at two large, urban, academic referral hospitals. PATIENTS: All consecutive adult patients admitted to the ICU from March 1, 2020, to June 7, 2020, with COVID-19 with clinical biomarkers and delirium assessments performed. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Daily concentrations of CRP, d-dimer, and ferritin were obtained. Coma (assessed by Richmond Agitation-Sedation Scale) and delirium (assessed by Confusion Assessment Method for the ICU/Confusion Assessment Method for the ICU-7) were measured bid. A cohort of 197 ICU patients with COVID-19 were included. Higher d-dimer (odds ratio [OR], 1.57; 95% CI, 1.17–2.12; p < 0.01) and ferritin quartiles (OR, 1.36; 95% CI, 1.02–1.81; p < 0.01) were associated with greater odds of the composite outcome of delirium/coma next day. d-dimer was associated with greater odds of next day delirium (OR, 1.49; 95% CI, 1.14–1.94; p < 0.01) and coma independently (OR, 1.52; 95% CI, 1.08–2.14; p = 0.017). Higher ferritin quartiles were associated with greater odds of next day delirium (OR, 1.33; 95% CI, 1.04–1.70; p = 0.026) and coma independently (OR, 1.59; 95% CI, 1.14–2.23; p < 0.01). Higher CRP quartiles were associated with coma (OR, 1.36; 95% CI, 1.03–1.79; p = 0.030) and delirium severity the next day (β = 0.30; se, 0.07; p ≤ 0.01). CONCLUSIONS: Our hypothesis-generating study found d-dimer and ferritin were associated with delirium/coma the following day, as well as delirium and coma independently. CRP was associated with next day coma and delirium severity. Larger studies to validate these results are needed.

Abstract IL-7 drives early B lymphopoiesis, but the underlying molecular circuits remain poorly understood, especially how Stat5-dependent and independent pathways contribute to this process. Combining transcriptome and proteome analyses and mouse genetic models, we show that IL-7 promotes anabolic metabolism and biosynthetic programs in pro-B cells. IL-7-mediated mTORC1 activation supported cell proliferation and metabolism in a Stat5-independent, Myc-dependent manner, but was largely dispensable for cell survival or Rag1 and Rag2 gene expression. mTORC1 was also required for Myc-driven lymphomagenesis. Importantly, PI3K and mTORC1 had discrete effects on Stat5 signaling and independently controlled B cell development. In fact, PI3K was actively suppressed by PTEN in pro-B cells to ensure proper IL-7R expression, Stat5 activation, heavy chain rearrangement and survival, suggesting the unexpected bifurcation of the classical PI3K-mTOR signaling. Altogether, our integrative analyses establish IL-7R-mTORC1-Myc and PTEN-mediated PI3K suppression as discrete signaling axes driving B cell development, with differential effects on IL-7R-Stat5 signaling.

Abstract Experimental autoimmune thyroiditis (EAT) is a model for Hashimoto’s thyroiditis which is characterized by mononuclear cell infiltration and follicular destruction. We previously reported on an Eα-transgenic strain (H2A−E+) that is highly susceptible to human (h) thyroglobulin (Tg)-induced EAT, but strongly tolerant to mouse (m)Tg. After mTg and adjuvant, traditional, resistant strains do not develop thyroiditis, nor detectable primed T cell proliferation, but do make anti-mTg Abs. By contrast, this H2Eb+ strain does not even produce mTg Abs. To identify what epitopes in the mouse thyroid hTg-primed, thyroiditogenic T cells recognize to evoke EAT, we first confirmed that hTg-primed cells do not respond to mTg, nor to other thyroid Ags. We next selected the three immunogenic epitopes on hTg known to be processed and presented by H2Eb, as the basis for synthesizing potential mTg epitopes. One 15-mer peptide, mTg409, did prime T cells, elicit Ab, and generate moderate thyroiditis. Thyroiditis incidence was increased after adoptive transfer of in vitro activated, mTg409-primed cells. Moreover, cells primed to hTg410, the corresponding, pathogenic hTg peptide, cross-reacted with mTg409, and vice versa. This mTg autoepitope, identified by using thyroiditogenic hTg epitopes, helps to explain the severe thyroiditis seen in this novel H2E+ transgenic model. (Supported by NIH grant DK45960)

Abstract Regulatory T (Treg) cell activation and expansion during neonatal life and in response to inflammation are critical for immunosuppression, yet the mechanisms governing these events are incompletely understood. We report that the oncogene and transcriptional regulator c-Myc (Myc) controls immune homeostasis through regulation of Treg cell accumulation and functional activation. Myc activity is enriched in Treg cells generated during neonatal life and responding to inflammation. Myc-deficient Treg cells show cell-intrinsic defects in overall accumulation and ability to transition to an activated state during early life or acute inflammation. Consequently, loss of Myc in Treg cells results in a rapid, early-onset autoimmune disorder accompanied by uncontrolled effector CD4+ and CD8+ T cell responses. We also provide evidence that Myc regulates mitochondrial oxidative metabolism but is dispensable for fatty acid oxidation (FAO). Indeed, Treg cell-specific deletion of Cox10, which is required for oxidative phosphorylation, but not Cpt1a, the rate-limiting enzyme for FAO, results in impaired Treg cell function and maturation. Thus, Myc coordinates Treg cell accumulation, transitional activation and metabolic programming to orchestrate immune homeostasis.

Abstract T cell anergy is a state of cellular hyporesponsiveness that prevents the propagation of an immune response to self. Consequently, the development and maintenance of the anergy program in T cells is tightly controlled. We found in wild type (WT) mice that anergic T cells, defined by co-expression of the cell surface markers CD73 and FR4, express lower expression of the transcription factor Runx1. To test if Runx1 acts as a negative regulator of the anergy program, we utilized a 1:1 mixed bone marrow chimera (BMC) system with ER-Cre Runx1 cKO and WT B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, ER-Cre Runx1 cKO CD4+ T cells develop a CD73+FR4+ anergic phenotype. This effect is cell-intrinsic since WT T cells from the same bone marrow chimera do not exhibit the anergic phenotype. Functionally, ER-Cre Runx1 cKO T cells fail to proliferate and produce less IL-2 after TCR/CD28 stimulation, consistent with anergy. Interestingly, WT B6.SJL T cells from the ER-Cre Runx1 cKO:B6.SJL BMC mice fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 additionally regulates a cell-extrinsic suppressive mechanism. Using depletion experiments, we found this extrinsic suppression requires FR4+ anergic T cells, but not CD25+ Tregs. Interestingly, the loss of Runx1 leads to the generation of CD73+FR4+ anergy in OT-II T cells, which do not engage their TCR. Thus, the loss of Runx1 drives anergy in the absence of TCR signaling. We conducted RNA-sequencing on ER-Cre Runx1 cKO and WT anergic T cells, and have identified several genes regulated by Runx1 that could play a role in anergy. Further analysis will serve to define the molecular mechanisms Runx1 employs to regulate T cell anergy.

Abstract The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on recent thymic emigrants, and leaving very few CD4+T cells in peripheral lymphoid organs. Interestingly, thymic- and splenic-derived T cells from CD4-Cre Runx1 cKO mice exhibit increased expression of markers indicative of anergy including FR4 and CD73, which is not seen in other genetically modified mice with a block in T cell maturation. These data suggest Runx1 plays a role in regulating the development of T cell anergy in CD4+T cells. We have generated a 1:1 mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre (ER-Cre) Runx1 conditional knockout and wild type B6.SJL bone marrow to delete Runx1 in mature CD4+ T cells. Upon tamoxifen treatment and deletion of Runx1, the ER-Cre Runx1 cKO CD4+ T cells spontaneously develop an anergic phenotype as they fail to proliferate upon TCR/CD28 stimulation, and co-express CD73 and FR4. This cell surface phenotype is cell intrinsic since wild type B6.SJL CD4+ T cells in the mixed bone marrow chimera do not upregulate CD73 and FR4 upon tamoxifen treatment. Surprisingly, the mixed BMC system reveals both the ER-Cre Runx1 cKO and the wild type B6.SJL peripheral T cells fail to proliferate in response to TCR/CD28 stimulation, suggesting Runx1 also regulates a cell-extrinsic suppressive mechanism controlling T cell tolerance. This cell-extrinsic induction of anergy requires Runx1-deficient CD4+ T cells, but it is not dependent on Runx1-deficient Tregs. Thus, Runx1 is a key regulator of T cell anergy, and further analysis of this BMC model will serve to uncover the mechanism.

Abstract The transcription factor Runx1 is critical for T cell development and maturation. CD4-Cre Runx1 conditional knockout mice have a block in T cell maturation, leading to complement deposition on CD4+ recent thymic emigrants, leaving very few CD4+ T cells in peripheral lymphoid organs. Consistent with a block in maturation, peripheral Runx1-deficient CD4+ T cells fail to produce normal levels of TNF-a upon stimulation. These CD4+ T cells that persist in CD4-Cre Runx1 cKO mice possess a phenotype not found in other genetically modified mice with a block in T cell maturation. Thymic- and splenic-derived T cells from the CD4-Cre Runx1 cKO exhibit altered expression of markers indicative of anergy including FR4 and CD73. This suggests Runx1 plays a role in regulating the development of T cell tolerance which is disrupted upon deletion of Runx1 in CD4+ T Cells. To test this, we have generated a mixed bone marrow chimera (BMC) system with Estrogen Receptor-Cre Runx1 knockout and B6.SJL bone marrow to knock out Runx1 in mature T cells. Surprisingly, in the mixed BMC both the ER-Cre Runx1 cKO and the B6.SJL peripheral T cells acquire a functionally hyporesponsive phenotype, suggesting that Runx1 regulates an extrinsic signaling mechanism controlling tolerance. Moving forward, this BMC model allows us to investigate the development of this anergic phenotype in mature T cells, and tests the hypothesis that Runx1 plays a critical role in regulating T cell tolerance.

Abstract The production of naïve B cells in bone marrow (BM) is critical for maintaining a diverse peripheral B cell pool to fight infection and establish lifelong immunity. The generation of naïve B cells is impaired in Flt3-ligand (FL-/-) mice contributing to reductions in splenic B cells. The goal of this study was to determine the consequence of FL-deficiency on splenic B cell maturation. We found significant reductions in numbers of transitional (TS) and follicular (FO), but not marginal zone (MZ) B cells in FL-/- mice. Mixed BM chimeras revealed that the reduction in TS B cells was cell extrinsic, linking microenvironmental signals to TS B cell maturation. FL administration into FL-/- mice restored the deficiency in TS B cells that do not express Flt3, suggesting that a FL responsive component of the microenvironment is critical for TS maturation. Intracellular Ki67 revealed a significant decrease in the proliferative capacity of TS cells in FL-/- mice. Expression of a Bcl2 transgene did not rescue TS cells, uncoupling FL-deficiency to Bcl2-regulated survival pathways. BAFF-R expression and serum levels of BAFF were comparable in WT and FL-/- mice, supporting BAFF-independent regulation of TS cells. These observations support an integral indirect role for Flt3 signaling in regulation of TS B cell maturation in the spleen.

Abstract Autoimmune uveitis affects 150,000 Americans annually and is a leading cause of blindness. Pathogenesis is thought to involve T cell mediated responses to retinal proteins, such as retinal arrestin (S-Ag). Susceptibility to autoimmune uveitis is strongly associated with HLA class I and class II, but relatively little is known about the nature of HLA class II alleles and about the epitopes that might be pathogenic in the context of different HLA class II molecules. Using a ‘humanized’ mouse model for experimental autoimmune uveitis (EAU) in HLA transgenic mice, we have identified permissive and non-permissive alleles of the HLA-DR and -DQ genes and characterized allele-specific uveitogenic epitopes. The sequences of these epitopes overlap with some of the previously identified peptides of S-Ag (M and N), which elicit memory responses in lymphocytes of uveitis patients. HLA-DR restricted S-Ag specific CD4+ T cells could be detected in blood and draining lymph nodes of immunized mice using HLA class II tetramers, demonstrating the potential use of this reagent to detect and isolate antigen specific T cells in uveitis patients. Our data provide new insights into the pathogenesis of human uveitis, and may help in the development of antigen specific therapies.

Abstract The COVID‐19 pandemic and its accompanying infection control measures introduced sudden and significant disruptions to the lives of children and adolescents around the world. Given the potential for negative impacts on the mental health of youths as a result of these changes, we conducted a systematic review and meta‐analysis to examine the prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances in children and adolescents during the pandemic. We searched major literature databases for relevant cross‐sectional or longitudinal studies that included primary and secondary school students or children and adolescents ≤18 years of age. Prevalence values were extracted, logit‐transformed, and pooled. Based on 191 included studies with 1,389,447 children and adolescents, we found the pooled prevalence of depressive symptoms, anxiety symptoms, and sleep disturbances to be 31%, 31%, and 42%, respectively. Age, grade levels, education levels, gender, geographical regions, and electronics use were correlated with the prevalence of mental health symptoms. The prevalence of mental health symptoms also increased with time, although signs of recovery and stabilization were also observed. Overall, the results from this review demonstrate the need for increased mental health research, monitoring, and intervention for children and adolescents during the current and future pandemics.