Multi-omic assessment shows dysregulation of pulmonary and systemic immunity to e-cigarette exposure.

Electronic cigarette (Ecig) use has become more common, gaining increasing acceptance as a safer alternative to tobacco smoking. However, the 2019 outbreak of Ecig and Vaping-Associated Lung Injury (EVALI) alerted the community to the potential for incorporation of deleterious ingredients such as vitamin E acetate into products without adequate safety testing. Understanding Ecig induced molecular changes in the lung and systemically can provide a path to safety assessment and protect consumers from unsafe formulations. While vitamin E acetate has been largely removed from commercial and illicit products, many Ecig products contain additives that remain largely uncharacterized. In this study, we determined the lung-specific effects as well as systemic immune effects in response to exposure to a common Ecig base, propylene glycol and vegetable glycerin (PGVG), with and without a 1% addition of phytol, a diterpene alcohol that has been found in commercial products. We exposed animals to PGVG with and without phytol and assessed metabolite, lipid, and transcriptional markers in the lung. We found both lung-specific as well as systemic effects in immune parameters, metabolites, and lipids. Phytol drove modest changes in lung function and increased splenic CD4 T cell populations. We also conducted multi-omic data integration to better understand early complex pulmonary responses, highlighting a central enhancement of acetylcholine responses and downregulation of palmitic acid connected with conventional flow cytometric assessments of lung, systemic inflammation, and pulmonary function. Our results demonstrate that Ecig exposure not only leads to changes in pulmonary function but also affects systemic immune and metabolic parameters.

CD43 sialoglycoprotein modulates cardiac inflammation and murine susceptibility to Trypanosoma cruzi infection.

CD43 (leukosialin) is a large sialoglycoprotein abundantly expressed on the surface of most cells from the hematopoietic lineage. CD43 is directly involved in the contact between cells participating in a series of events such as signaling, adherence and host parasite interactions. In this study we examined the role of CD43 in the immune response against Trypanosoma cruzi, the protozoan parasite that causes Chagas' disease, a potential life-threatening illness endemic in 21 Latin American countries according to the WHO. The acute stage of infection is marked by intense parasitemia and cardiac tissue parasitism, resulting in the recruitment of inflammatory cells and acute damage to the heart tissue. We show here that CD43-/- mice were more resistant to infection due to increased cytotoxicity of antigen specific CD8+ T cells and reduced inflammatory infiltration in the cardiac tissue, both contributing to lower cardiomyocyte damage. In addition, we demonstrate that the induction of acute myocarditis involves the engagement of CD43 cytoplasmic tripeptide sequence KRR to ezrin-radixin-moiesin cytoskeletal proteins. Together, our results show the participation of CD43 in different events involved in the pathogenesis of T. cruzi infection, contributing to a better overall understanding of the mechanisms underlying the pathogenesis of acute chagasic cardiomyopathy.

Epigenetic silencing of SOCS5 potentiates JAK-STAT signaling and progression of T-cell acute lymphoblastic leukemia.

Activating mutations in cytokine receptors and transcriptional regulators govern aberrant signal transduction in T-cell lineage acute lymphoblastic leukemia (T-ALL). However, the roles played by suppressors of cytokine signaling remain incompletely understood. We examined the regulatory roles of suppressor of cytokine signaling 5 (SOCS5) in T-ALL cellular signaling networks and leukemia progression. We found that SOCS5 was differentially expressed in primary T-ALL and its expression levels were lowered in HOXA-deregulated leukemia harboring KMT2A gene rearrangements. Here, we report that SOCS5 expression is epigenetically regulated by DNA methyltransferase-3A-mediated DNA methylation and methyl CpG binding protein-2-mediated histone deacetylation. We show that SOCS5 negatively regulates T-ALL cell growth and cell cycle progression but has no effect on apoptotic cell death. Mechanistically, SOCS5 silencing induces activation of JAK-STAT signaling, and negatively regulates interleukin-7 and interleukin-4 receptors. Using a human T-ALL murine xenograft model, we show that genetic inactivation of SOCS5 accelerates leukemia engraftment and progression, and leukemia burden. We postulate that SOCS5 is epigenetically deregulated in T-ALL and serves as an important regulator of T-ALL cell proliferation and leukemic progression. Our results link aberrant downregulation of SOCS5 expression to the enhanced activation of the JAK-STAT and cytokine receptor-signaling cascade in T-ALL.

Genetically enhancing the expression of chemokine domain of CX3CL1 fails to prevent tau pathology in mouse models of tauopathy.

BACKGROUND: Fractalkine (CX3CL1) and its receptor (CX3CR1) play an important role in regulating microglial function. We have previously shown that Cx3cr1 deficiency exacerbated tau pathology and led to cognitive impairment. However, it is still unclear if the chemokine domain of the ligand CX3CL1 is essential in regulating neuronal tau pathology. METHODS: We used transgenic mice lacking endogenous Cx3cl1 (Cx3cl1-/-) and expressing only obligatory soluble form (with only chemokine domain) and lacking the mucin stalk of CX3CL1 (referred to as Cx3cl1105Δ mice) to assess tau pathology and behavioral function in both lipopolysaccharide (LPS) and genetic (hTau) mouse models of tauopathy. RESULTS: First, increased basal tau levels accompanied microglial activation in Cx3cl1105Δ mice compared to control groups. Second, increased CD45+ and F4/80+ neuroinflammation and tau phosphorylation were observed in LPS, hTau/Cx3cl1-/-, and hTau/Cx3cl1105Δ mouse models of tau pathology, which correlated with impaired spatial learning. Finally, microglial cell surface expression of CX3CR1 was reduced in Cx3cl1105Δ mice, suggesting enhanced fractalkine receptor internalization (mimicking Cx3cr1 deletion), which likely contributes to the elevated tau pathology. CONCLUSIONS: Collectively, our data suggest that overexpression of only chemokine domain of CX3CL1 does not protect against tau pathology.

Central role of T helper 17 cells in chronic hypoxia-induced pulmonary hypertension.

Inflammation is a prominent pathological feature in pulmonary arterial hypertension, as demonstrated by pulmonary vascular infiltration of inflammatory cells, including T and B lymphocytes. However, the contribution of the adaptive immune system is not well characterized in pulmonary hypertension caused by chronic hypoxia. CD4+ T cells are required for initiating and maintaining inflammation, suggesting that these cells could play an important role in the pathogenesis of hypoxic pulmonary hypertension. Our objective was to test the hypothesis that CD4+ T cells, specifically the T helper 17 subset, contribute to chronic hypoxia-induced pulmonary hypertension. We compared indices of pulmonary hypertension resulting from chronic hypoxia (3 wk) in wild-type mice and recombination-activating gene 1 knockout mice (RAG1-/-, lacking mature T and B cells). Separate sets of mice were adoptively transferred with CD4+, CD8+, or T helper 17 cells before normoxic or chronic hypoxic exposure to evaluate the involvement of specific T cell subsets. RAG1-/- mice had diminished right ventricular systolic pressure and arterial remodeling compared with wild-type mice exposed to chronic hypoxia. Adoptive transfer of CD4+ but not CD8+ T cells restored the hypertensive phenotype in RAG1-/- mice. Interestingly, RAG1-/- mice receiving T helper 17 cells displayed evidence of pulmonary hypertension independent of chronic hypoxia. Supporting our hypothesis, depletion of CD4+ cells or treatment with SR1001, an inhibitor of T helper 17 cell development, prevented increased pressure and remodeling responses to chronic hypoxia. We conclude that T helper 17 cells play a key role in the development of chronic hypoxia-induced pulmonary hypertension.

Self-Contained Statistical Analysis of Gene Sets.

Microarrays are a powerful tool for studying differential gene expression. However, lists of many differentially expressed genes are often generated, and unraveling meaningful biological processes from the lists can be challenging. For this reason, investigators have sought to quantify the statistical probability of compiled gene sets rather than individual genes. The gene sets typically are organized around a biological theme or pathway. We compute correlations between different gene set tests and elect to use Fisher's self-contained method for gene set analysis. We improve Fisher's differential expression analysis of a gene set by limiting the p-value of an individual gene within the gene set to prevent a small percentage of genes from determining the statistical significance of the entire set. In addition, we also compute dependencies among genes within the set to determine which genes are statistically linked. The method is applied to T-ALL (T-lineage Acute Lymphoblastic Leukemia) to identify differentially expressed gene sets between T-ALL and normal patients and T-ALL and AML (Acute Myeloid Leukemia) patients.

ICOS-expressing lymphocytes promote resolution of CD8-mediated lung injury in a mouse model of lung rejection.

Acute rejection, a common complication of lung transplantation, may promote obliterative bronchiolitis leading to graft failure in lung transplant recipients. During acute rejection episodes, CD8(+) T cells can contribute to lung epithelial injury but the mechanisms promoting and controlling CD8-mediated injury in the lung are not well understood. To study the mechanisms regulating CD8(+) T cell-mediated lung rejection, we used a transgenic model in which adoptively transferred ovalbumin (OVA)-specific cytotoxic T lymphocytes (CTL) induce lung injury in mice expressing an ovalbumin transgene in the small airway epithelium of the lungs (CC10-OVA mice). The lung pathology is similar to findings in humans with acute lung transplant. In the presence of an intact immune response the inflammation resolves by day 30. Using CC10-OVA.RAG(-/-) mice, we found that CD4(+) T cells and ICOS(+/+) T cells were required for protection against lethal lung injury, while neutrophil depletion was not protective. In addition, CD4(+)Foxp3 (+) ICOS(+) T cells were enriched in the lungs of animals surviving lung injury and ICOS(+/+) Tregs promoted survival in animals that received ICOS(-/-) T cells. Direct comparison of ICOS(-/-) Tregs to ICOS(+/+) Tregs found defects in vitro but no differences in the ability of ICOS(-/-) Tregs to protect from lethal lung injury. These data suggest that ICOS affects Treg development but is not necessarily required for Treg effector function.

CD43-mediated IFN-γ production by CD8+ T cells promotes abdominal aortic aneurysm in mice.

CD43 is a glycosylated surface protein abundantly expressed on lymphocytes. Its role in immune responses has been difficult to clearly establish, with evidence supporting both costimulatory and inhibitory functions. In addition, its contribution to disease pathogenesis remains elusive. Using a well-characterized murine model of elastase-induced abdominal aortic aneurysm (AAA) that recapitulates many key features of the human disease, we established that the presence of CD43 on T cells is required for AAA formation. Moreover, we found that IFN-γ-producing CD8(+) T cells, but not CD4(+) T cells, promote the development of aneurysm by enhancing cellular apoptosis and matrix metalloprotease activity. Reconstitution with IFN-γ-producing CD8(+) T cells or recombinant IFN-γ promotes the aneurysm phenotype in CD43(-/-) mice, whereas IFN-γ antagonism abrogates disease in wild-type animals. Furthermore, we showed that the presence of CD43 with an intact cytoplasmic domain capable of binding to ezrin-radixin-moesin cytoskeletal proteins is essential for optimal in vivo IFN-γ production by T cells and aneurysm formation. We have thus identified a robust physiologic role for CD43 in a relevant animal model and established an important in vivo function for CD43-dependent regulation of IFN-γ production. These results further suggest that IFN-γ antagonism or selective blockade of CD43(+)CD8(+) T cell activities merits further investigation for immunotherapy in AAA.

Beta-catenin inhibits T cell activation by selective interference with linker for activation of T cells-phospholipase C-γ1 phosphorylation.

Despite the defined function of the ß-catenin pathway in thymocytes, its functional role in peripheral T cells is poorly understood. We report that in a mouse model, ß-catenin protein is constitutively degraded in peripheral T cells. Introduction of stabilized ß-catenin into primary T cells inhibited proliferation and cytokine secretion after TCR stimulation and blunted effector cell differentiation. Functional and biochemical studies revealed that ß-catenin selectively inhibited linker for activation of T cells phosphorylation on tyrosine 136, which was associated with defective phospholipase C-γ1 phosphorylation and calcium signaling but normal ERK activation. Our findings indicate that ß-catenin negatively regulates T cell activation by a previously undescribed mechanism and suggest that conditions under which ß-catenin might be inducibly stabilized in vivo would be inhibitory for T cell-based immunity.

Ezrin is highly expressed in early thymocytes, but dispensable for T cell development in mice.

BACKGROUND: Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/-) mice likely arise as a consequence of nutritional stress. CONCLUSIONS/SIGNIFICANCE: We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.

Differential roles for Wiskott-Aldrich syndrome protein in immune synapse formation and IL-2 production.

Wiskott-Aldrich syndrome protein (WASP)-deficient T cells exhibit defects in IL-2 production that are widely believed to stem from primary defects in actin remodeling and immune synapse formation. Surprisingly, however, we find that WASP-deficient T cells responding to Ag-specific APCs polymerize actin and organize talin and PKC theta normally, forming an immune synapse that is stable for at least 3 h. At low doses of peptide, WASP-deficient T cells show less efficient talin and PKC theta polarization. Thus, although WASP may facilitate immune synapse formation at low peptide concentrations, WASP is not required for this process. Defects in IL-2 production are observed even under conditions in which immune synapse formation proceeds normally, suggesting that the role of WASP in regulating IL-2 production is independent of its role in immune synapse formation.

SLP-76 coordinates Nck-dependent Wiskott-Aldrich syndrome protein recruitment with Vav-1/Cdc42-dependent Wiskott-Aldrich syndrome protein activation at the T cell-APC contact site.

We have shown previously that Wiskott-Aldrich syndrome protein (WASP) activation at the site of T cell-APC interaction is a two-step process, with recruitment dependent on the proline-rich domain and activation dependent on binding of Cdc42-GTP to the GTPase binding domain. Here, we show that WASP recruitment occurs through binding to the C-terminal Src homology 3 domain of Nck. In contrast, WASP activation requires Vav-1. In Vav-1-deficient T cells, WASP recruitment proceeds normally, but localized activation of Cdc42 and WASP is disrupted. The recruitment and activation of WASP are coordinated by tyrosine-phosphorylated Src homology 2 domain-containing leukocyte protein of 76 kDa, which functions as a scaffold, bringing Nck and WASP into proximity with Vav-1 and Cdc42-GTP. Taken together, these findings reconstruct the signaling pathway leading from TCR ligation to localized WASP activation.