Blake Frey

Constitutive androstane receptor (CAR, encoded by Nr1i3) is a nuclear xenobiotic receptor that mediates hepatic drug and bile acid metabolism. We previously identified CAR as also operating in CD4 T helper (Th) cells, where CAR dependent gene expression mitigates bile acid toxicity and promotes a Foxp3 negative IL10 positive type 1 regulatory (Tr1) like phenotype in the small intestine. Here, we show that CAR acts early and specifically during the priming of type 1 immune responses to stabilize Tr1 lineage commitment. CAR dependent Tr1 cells formed during type 1 (Th1 associated) but not type 3 (Th17 associated) intestinal inflammation. In vitro, IL27 upregulated CAR expression during naive Th cell activation, which contributed to Il10 induction. Single cell analysis revealed that naive Th cells primed with IL27 adopt a multipotent Th1/Tr1 precursor (THR1p) transcriptional state, which subsequently diverges into Th1 or Tr1 developmental trajectories. CAR transcriptional activity peaked in THR1p cells, upregulating Tr1 genes, including Il10, and repressing Th1 genes. Moreover, glucocorticoid receptor activation, which increases CAR expression in hepatocytes, synergized with IL27 to augment both CAR expression and CAR dependent Tr1 differentiation. Together, these results suggest that CAR acts in a lineage biased manner to enforce Tr1 mediated immune tolerance during type 1 intestinal inflammation, and this pathway is amplified by glucocorticoids.

Hallmark findings in age-related macular degeneration (AMD) include the accumulation of extracellular lipid and vasodegeneration of the choriocapillaris. Choroidal inflammation has long been associated with AMD, but little is known about the immune landscape of the human choroid. Using 3D multiplex immunofluorescence, single-cell RNA sequencing, and flow cytometry, we unravel the cellular composition and spatial organization of the human choroid and the immune cells within it. We identify two populations of choroidal macrophages with distinct FOLR2 expression that account for the majority of myeloid cells. FOLR2+ macrophages predominate in the nondiseased eye, express lipid-handling machinery, uptake lipoprotein particles, and contain high amounts of lipid. In AMD, FOLR2+ macrophages are decreased in number and exhibit dysfunctional lipoprotein metabolism. In mice, FOLR2+ macrophages are negative for the postnatal fate-reporter Ms4a3, and their depletion causes an accelerated AMD-like phenotype. Our results show that prenatally derived resident macrophages decline in AMD and are implicated in multiple hallmark functions known to be compromised in the disease.

Background. Racial disparities are well described in glycemic outcomes in youth with Type 1 diabetes mellites (T1D). Hemoglobin A1c (HbA1c) has some limitations in comparing glycemia across patient groups as there are individual variations in mean glucose and HbA1c. Objective. This study aimed to compare glycemic metrics obtained from (Dexcom G6) continuous glucose monitor (CGM) device with HbA1c levels controlling for race, age, duration of diabetes, race, insurance status, and insulin pump use with glycemic control. Subjects and Methods. Data analyzed included 188 patients, majority non-Hispanic White (NHW) (n = 147, 78.2%) and majority privately insured (n = 147, 78.2%). Half of the patients were using insulin pumps, (n = 94, 50.0%) and approximately half were female. Median age was 16.6 (interquartile range: 14.2–18.2) years old with a median age of diabetes diagnosis at 9.3-years old. Results. Significant differences were observed between NHW and non-Hispanic Black (NHB) patients in terms of HbA1c, 90-day mean glucose, and 90-day time >250 mg/dL (>13.9 mmol/L) (7.6% vs. 9.2%, 181 mg/dL vs. 220 mg/dL, and 16.3% vs. 34.7%, respectively, for all comparisons). Multiple linear regression analysis was performed to predict the influence of age, duration of diabetes, race, insurance status, and insulin administration on glycemic outcomes. Regression analysis revealed significant equations for all glycemic outcomes, demonstrating a strong correlation ( , , and , respectively). However, after controlling for these variables, only race and duration of diabetes remained independently associated with glycemic outcomes, suggesting that these factors strongly influence glycemic control independent of age, sex, insurance, and pump use. Conclusion. Even in a subset of youth with T1D using CGM with high rates of insulin pump use, disparities in glycemic outcomes persist. When evaluating glycemic outcomes, race remained a significant cofactor despite controlling for age, duration of diabetes, sex, insurance status, and insulin administration type. These results add to the existing literature, and demonstrate race remains strong predictor of glycemic outcomes.

Several independent lines of evidence suggest that megakaryocytes are dysfunctional in severe COVID-19. Herein, we characterized peripheral circulating megakaryocytes in a large cohort of inpatients with COVID-19 and correlated the subpopulation frequencies with clinical outcomes. Using peripheral blood, we show that megakaryocytes are increased in the systemic circulation in COVID-19, and we identify and validate S100A8/A9 as a defining marker of megakaryocyte dysfunction. We further reveal a subpopulation of S100A8/A9+ megakaryocytes that contain severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein and RNA. Using flow cytometry of peripheral blood and in vitro studies on SARS-CoV-2–infected primary human megakaryocytes, we demonstrate that megakaryocytes can transfer viral antigens to emerging platelets. Mechanistically, we show that SARS-CoV-2–containing megakaryocytes are nuclear factor κB (NF-κB)-activated, via p65 and p52; express the NF-κB–mediated cytokines interleukin-6 (IL-6) and IL-1β; and display high surface expression of Toll-like receptor 2 (TLR2) and TLR4, canonical drivers of NF-κB. In a cohort of 218 inpatients with COVID-19, we correlate frequencies of megakaryocyte subpopulations with clinical outcomes and show that SARS-CoV-2–containing megakaryocytes are a strong risk factor for mortality and multiorgan injury, including respiratory failure, mechanical ventilation, acute kidney injury, thrombotic events, and intensive care unit admission. Furthermore, we show that SARS-CoV-2+ megakaryocytes are present in lung and brain autopsy tissues from deceased donors who had COVID-19. To our knowledge, this study offers the first evidence implicating SARS-CoV-2+ peripheral megakaryocytes in severe disease and suggests that circulating megakaryocytes warrant investigation in inflammatory disorders beyond COVID-19.

Bile acids (BAs) are lipid emulsifying metabolites synthesized in hepatocytes and maintained in vivo through enterohepatic circulation between the liver and small intestine ¹ . As detergents, BAs can cause toxicity and inflammation in enterohepatic tissues ² . Nuclear receptors maintain BA homeostasis in hepatocytes and enterocytes ³ , but it is unclear how mucosal immune cells tolerate high BA concentrations in the small intestine lamina propria (siLP). We previously reported that CD4 ⁺ T effector (Teff) cells upregulate expression of the xenobiotic transporter MDR1/ ABCB1 in the siLP to prevent BA toxicity and suppress Crohn’s disease-like small bowel inflammation ⁴ . Here, we identify the nuclear xenobiotic receptor, constitutive androstane receptor (CAR/ NR1I3 ), as a regulator of MDR1 expression in T cells, and safeguard against BA toxicity and inflammation in the small intestine. CAR was activated and induced large-scale transcriptional reprograming in Teff cells infiltrating the siLP, but not the colon. CAR induced expression of detoxifying enzymes and transporters in siLP Teff cells, as in hepatocytes, but also the key anti-inflammatory cytokine, Il10 . Accordingly, CAR-deficiency in T cells exacerbated, whereas pharmacologic CAR activation suppressed, BA-driven ileitis in T cell-reconstituted Rag ^−/− mice. These data suggest that CAR acts locally in small intestinal T cells to detoxify BAs and resolve inflammation. Activation of this program offers an unexpected strategy to treat small bowel Crohn’s disease, and provides evidence of lymphocyte sub-specialization within the small intestine.

There are no defined correlates of protection for any intracellular pathogen, including the bacterium Francisella tularensis, which causes tularemia. Evaluating vaccine efficacy against sporadic diseases like tularemia using field trials is problematic, and therefore alternative strategies to test vaccine candidates like the Francisella Live Vaccine Strain (LVS), such as testing in animals and applying correlate measurements, are needed. Recently, we described a promising correlate strategy that predicted the degree of vaccine-induced protection in mice given parenteral challenges, primarily when using an attenuated Francisella strain. Here, we demonstrate that using peripheral blood lymphocytes (PBLs) in this approach predicts LVS-mediated protection against respiratory challenge of Fischer 344 rats with fully virulent F. tularensis, with exceptional sensitivity and specificity. Rats were vaccinated with a panel of LVS-derived vaccines and subsequently given lethal respiratory challenges with Type A F. tularensis. In parallel, PBLs from vaccinated rats were evaluated for their functional ability to control intramacrophage Francisella growth in in vitro co-culture assays. PBLs recovered from co-cultures were also evaluated for relative gene expression using a large panel of genes identified in murine studies. In vitro control of LVS intramacrophage replication reflected the hierarchy of protection. Further, despite variability between individuals, 22 genes were significantly more up-regulated in PBLs from rats vaccinated with LVS compared to those from rats vaccinated with the variant LVS-R or heat-killed LVS, which were poorly protective. These genes included IFN-γ, IL-21, NOS2, LTA, T-bet, IL-12rβ2, and CCL5. Most importantly, combining quantifications of intramacrophage growth control with 5–7 gene expression levels using multivariate analyses discriminated protected from non-protected individuals with greater than 95% sensitivity and specificity. The results therefore support translation of this approach to non-human primates and people to evaluate new vaccines against Francisella and other intracellular pathogens.

Unlike cytosolic processing and presentation of viral Ags by virus-infected cells, Ags first expressed in infected nonprofessional APCs, such as CD4⁺ T cells in the case of HIV, are taken up by dendritic cells and cross-presented. This generally requires entry through the endocytic pathway, where endosomal proteases have first access for processing. Thus, understanding virus escape during cross-presentation requires an understanding of resistance to endosomal proteases, such as cathepsin S (CatS). We have modified HIV-1_MN gp120 by mutating a key CatS cleavage site (Thr³²²Thr³²³) in the V3 loop of the immunodominant epitope IGPGRAFYTT to IGPGRAFYVV to prevent digestion. We found this mutation to facilitate cross-presentation and provide evidence from MHC binding and X-ray crystallographic structural studies that this results from preservation of the epitope rather than an increased epitope affinity for the MHC class I molecule. In contrast, when the protein is expressed by a vaccinia virus in the cytosol, the wild-type protein is immunogenic without this mutation. These proof-of-concept results show that a virus like HIV, infecting predominantly nonprofessional presenting cells, can escape T cell recognition by incorporating a CatS cleavage site that leads to destruction of an immunodominant epitope when the Ag undergoes endosomal cross-presentation.

Myeloid derived suppressor cells (MDSCs), which suppress anti-tumor or anti-viral immune responses, are expanded in the peripheral blood and tissues of patients/animals with cancer or viral infectious diseases. We here show that in chronic SIV infection of Indian rhesus macaques, the frequency of MDSCs in the bone marrow (BM) was paradoxically and unexpectedly decreased, but increased in peripheral blood. Reduction of BM MDSCs was found in both CD14⁺MDSC and Lin^-CD15⁺MDSC subsets. The reduction of MDSCs correlated with high plasma viral loads and low CD4⁺ T cell counts, suggesting that depletion of BM MDSCs was associated with SIV/AIDS disease progression. Of note, in SHIV_SF162P4-infected macaques, which naturally control viral replication within a few months of infection, the frequency of MDSCs in the bone marrow was unchanged. To investigate the mechanisms by which BM MDSCs were reduced during chronic SIV infection, we tested several hypotheses: depletion due to viral infection, alterations in MDSC trafficking, and/or poor MDSC replenishment. We found that the possible mobilization of MDSCs from BM to peripheral tissues and the slow self-replenishment of MDSCs in the BM, along with the viral infection-induced depletion, all contribute to the observed BM MDSC reduction. We first demonstrate MDSC SIV infection in vivo. Correlation between BM CD14⁺MDSC reduction and CD8⁺ T cell activation in tissues is consistent with decreased immune suppression by MDSCs. Thus, depletion of BM MDSCs may contribute to the pathologic immune activation during chronic SIV infection and by extension HIV infection. Both cancer and infectious diseases including HIV/AIDS lead to the accumulation of myeloid-derived suppressor cells (MDSCs), which can effectively suppress anti-tumor and anti-viral T cell responses to dampen protective immunity. Using a macaque model, we found unexpectedly that the MDSCs in bone marrow (BM) decreased after chronic simian immunodeficiency virus (SIV) infection compared with healthy controls. This was in sharp contrast to the general increase of MDSCs observed in BM during cancer and other infectious/inflammatory diseases, and also contrary to the MDSC expansion in HIV/SIV-infected PBMCs. We further demonstrated that the loss of MDSCs in the bone marrow was associated with the progression to AIDS disease. Investigating the mechanisms by which the MDSCs were decreased in the SIV-infected bone marrow, we found that the possible mobilization of MDSCs from bone marrow to peripheral tissues and the slow self-replenishment of MDSCs in the bone marrow, along with the viral infection-induced depletion, all contribute to the observed bone marrow MDSC reduction. Indeed, this is the first demonstration to our knowledge of SIV infection of MDSCs in vivo. Because of the suppressive nature of the MDSCs, the CD8⁺ T cells might not be effective in killing the virally infected MDSCs. It is tempting to speculate that MDSCs may constitute latent reservoirs. Overall, our data showed that MDSCs act as a double-edged sword in HIV/SIV-infection, and the decrease of MDSCs in bone marrow after SIV infection could serve as an indicator of immune regulatory exhaustion and also contribute to the observed immune hyperactivation seen in HIV/AIDS.

The programmed death-1 receptor is expressed on a wide range of immune effector cells, including T cells, natural killer T cells, dendritic cells, macrophages, and natural killer cells. In malignancies and chronic viral infections, increased expression of programmed death-1 by T cells is generally associated with a poor prognosis. However, its role in early host microbial defense at the intestinal mucosa is not well understood. We report that programmed death-1 expression is increased on conventional natural killer cells but not on CD4⁺, CD8⁺ or natural killer T cells, or CD11b⁺ or CD11c⁺ macrophages or dendritic cells after infection with the mouse pathogen Citrobacter rodentium. Mice genetically deficient in programmed death-1 or treated with anti–programmed death-1 antibody were more susceptible to acute enteric and systemic infection with Citrobacter rodentium. Wild-type but not programmed death-1–deficient mice infected with Citrobacter rodentium showed significantly increased expression of the conventional mucosal NK cell effector molecules granzyme B and perforin. In contrast, natural killer cells from programmed death-1–deficient mice had impaired expression of those mediators. Consistent with programmed death-1 being important for intracellular expression of natural killer cell effector molecules, mice depleted of natural killer cells and perforin-deficient mice manifested increased susceptibility to acute enteric infection with Citrobacter rodentium. Our findings suggest that increased programmed death-1 signaling pathway expression by conventional natural killer cells promotes host protection at the intestinal mucosa during acute infection with a bacterial gut pathogen by enhancing the expression and production of important effectors of natural killer cell function.