Disruption of a self-amplifying catecholamine loop reduces cytokine release syndrome.

Cytokine release syndrome (CRS) is a life-threatening complication of several new immunotherapies used to treat cancers and autoimmune diseases1-5. Here we report that atrial natriuretic peptide can protect mice from CRS induced by such agents by reducing the levels of circulating catecholamines. Catecholamines were found to orchestrate an immunodysregulation resulting from oncolytic bacteria and lipopolysaccharide through a self-amplifying loop in macrophages. Myeloid-specific deletion of tyrosine hydroxylase inhibited this circuit. Cytokine release induced by T-cell-activating therapeutic agents was also accompanied by a catecholamine surge and inhibition of catecholamine synthesis reduced cytokine release in vitro and in mice. Pharmacologic catecholamine blockade with metyrosine protected mice from lethal complications of CRS resulting from infections and various biotherapeutic agents including oncolytic bacteria, T-cell-targeting antibodies and CAR-T cells. Our study identifies catecholamines as an essential component of the cytokine release that can be modulated by specific blockers without impairing the therapeutic response.

Intestinal helminths regulate lethal acute graft-versus-host disease and preserve the graft-versus-tumor effect in mice.

Donor T lymphocyte transfer with hematopoietic stem cells suppresses residual tumor growth (graft-versus-tumor [GVT]) in cancer patients undergoing bone marrow transplantation (BMT). However, donor T cell reactivity to host organs causes severe and potentially lethal inflammation called graft-versus-host disease (GVHD). High-dose steroids or other immunosuppressive drugs are used to treat GVHD that have limited ability to control the inflammation while incurring long-term toxicity. Novel strategies are needed to modulate GVHD, preserve GVT, and improve the outcome of BMT. Regulatory T cells (Tregs) control alloantigen-sensitized inflammation of GVHD, sustain GVT, and prevent mortality in BMT. Helminths colonizing the alimentary tract dramatically increase the Treg activity, thereby modulating intestinal or systemic inflammatory responses. These observations led us to hypothesize that helminths can regulate GVHD and maintain GVT in mice. Acute GVHD was induced in helminth (Heligmosomoides polygyrus)-infected or uninfected BALB/c recipients of C57BL/6 donor grafts. Helminth infection suppressed donor T cell inflammatory cytokine generation and reduced GVHD-related mortality, but maintained GVT. H. polygyrus colonization promoted the survival of TGF-ß-generating recipient Tregs after a conditioning regimen with total body irradiation and led to a TGF-ß-dependent in vivo expansion/maturation of donor Tregs after BMT. Helminths did not control GVHD when T cells unresponsive to TGF-ß-mediated immune regulation were used as donor T lymphocytes. These results suggest that helminths suppress acute GVHD using Tregs and TGF-ß-dependent pathways in mice. Helminthic regulation of GVHD and GVT through intestinal immune conditioning may improve the outcome of BMT.

Tim-3 directly enhances CD8 T cell responses to acute Listeria monocytogenes infection.

T cell Ig and mucin domain (Tim) 3 is a surface molecule expressed throughout the immune system that can mediate both stimulatory and inhibitory effects. Previous studies have provided evidence that Tim-3 functions to enforce CD8 T cell exhaustion, a dysfunctional state associated with chronic stimulation. In contrast, the role of Tim-3 in the regulation of CD8 T cell responses to acute and transient stimulation remains undefined. To address this knowledge gap, we examined how Tim-3 affects CD8 T cell responses to acute Listeria monocytogenes infection. Analysis of wild-type (WT) mice infected with L. monocytogenes revealed that Tim-3 was transiently expressed by activated CD8 T cells and was associated primarily with acquisition of an effector phenotype. Comparison of responses to L. monocytogenes by WT and Tim-3 knockout (KO) mice showed that the absence of Tim-3 significantly reduced the magnitudes of both primary and secondary CD8 T cell responses, which correlated with decreased IFN-γ production and degranulation by Tim-3 KO cells stimulated with peptide Ag ex vivo. To address the T cell-intrinsic role of Tim-3, we analyzed responses to L. monocytogenes infection by WT and Tim-3 KO TCR-transgenic CD8 T cells following adoptive transfer into a shared WT host. In this setting, the accumulation of CD8 T cells and the generation of cytokine-producing cells were significantly reduced by the lack of Tim-3, demonstrating that this molecule has a direct effect on CD8 T cell function. Combined, our results suggest that Tim-3 can mediate a stimulatory effect on CD8 T cell responses to an acute infection.

NFIL3-deficient mice develop microbiota-dependent, IL-12/23-driven spontaneous colitis.

NFIL3 is a transcription factor that regulates multiple immunologic functions. In myeloid cells, NFIL3 is IL-10 inducible and has a key role as a repressor of IL-12p40 transcription. NFIL3 is a susceptibility gene for the human inflammatory bowel diseases. In this article, we describe spontaneous colitis in Nfil3(-/-) mice. Mice lacking both Nfil3 and Il10 had severe early-onset colitis, suggesting that NFIL3 and IL-10 independently regulate mucosal homeostasis. Lymphocytes were necessary for colitis, because Nfil3/Rag1 double-knockout mice were protected from disease. However, Nfil3/Rag1 double-knockout mice adoptively transferred with wild-type CD4(+) T cells developed severe colitis compared with Rag1(-/-) recipients, suggesting that colitis was linked to defects in innate immune cells. Colitis was abrogated in Nfil3/Il12b double-deficient mice, identifying Il12b dysregulation as a central pathogenic event. Finally, germ-free Nfil3(-/-) mice do not develop colonic inflammation. Thus, NFIL3 is a microbiota-dependent, IL-10-independent regulator of mucosal homeostasis via IL-12p40.

NFIL3/E4BP4 controls type 2 T helper cell cytokine expression.

Type 2 T helper (T(H)2) cells are critical for the development of allergic immune responses; however, the molecular mechanism controlling their effector function is still largely unclear. Here, we report that the transcription factor NFIL3/E4BP4 regulates cytokine production and effector function by T(H)2 cells. NFIL3 is highly expressed in T(H)2 cells but much less in T(H)1 cells. Production of interleukin (IL)-13 and IL-5 is significantly increased in Nfil3(-/-) T(H)2 cells and is decreased by expression of NFIL3 in wild-type T(H)2 cells. NFIL3 directly binds to and negatively regulates the Il13 gene. In contrast, IL-4 production is decreased in Nfil3(-/-) T(H)2 cells. Increased IL-13 and IL-5 together with decreased IL-4 production by antigen-stimulated splenocytes from the immunized Nfil3(-/-) mice was also observed. The ability of NFIL3 to alter T(H)2 cytokine production is a T-cell intrinsic effect. Taken together, these data indicate that NFIL3 is a key regulator of T(H)2 responses.

The Johns Hopkins Physician-Scientist Training Program to Enhance Institutional Retention and Entry Into Academic Positions: An Evaluation of Program Effectiveness and Outcomes.

PURPOSE: The Johns Hopkins Physician-Scientist Training Program (PSTP) was implemented to overcome well-documented challenges in training and retaining physician-scientists by providing physician-scientist pathway training for residents and clinical fellows. The program's core tenets include monthly seminars, individualized feedback on project proposals, access to mentors, and institutional funding opportunities. This study evaluated the effectiveness and outcomes of the PTSP and provides a framework for replication. METHOD: A query of institutional demographic data and bibliometric variables of the PSTP participants (2017-2020) at a single academic medical center was conducted in 2021. In addition, a voluntary survey collected personal and program evaluation information. RESULTS: Of 145 PSTP scholars, 59 (41%) were women, and 41 (31%), 8 (6%), and 6 (5%) of scholars self-identified as Asian, Hispanic, and Black, respectively. Thirty-three (23%) scholars received PSTP research support or career development microgrants. Of 66 PSTP graduates, 29 (44%) remained at Johns Hopkins as clinical fellows or faculty. Of 48 PSTP graduates in a post-training position, 42 (88%) were in academia, with the majority, 29 (76%), holding the rank of assistant professor. Fifty-nine of 140 available participants responded to the survey (42% response rate). The top-cited reason for joining the PSTP was exposure to mentors and administration (50/58 respondents, 86%), followed by seeking scholarly opportunities (37/58 respondents, 64%). Most scholars intended to continue a career as a physician-scientist. CONCLUSIONS: The PSTP provides internal research support and institutional oversight. Although establishing close mentor-mentee relationships requires individualized approaches, the PSTP provided structured academic pathways that enhanced participating scholars' ability to apply for grants and jobs. The vast majority continued their careers as physician-scientists after training. In light of the national evidence of a "leaky physician-scientist pipeline," programs such as the PSTP can be critical to entry into early academic career positions and institutional retention.

Tim-1 regulates Th2 responses in an airway hypersensitivity model.

T-cell immunoglobulin mucin-1 (Tim-1) is a transmembrane protein postulated to be a key regulator of Th2-type immune responses. This hypothesis is based in part upon genetic studies associating Tim-1 polymorphisms in mice with a bias toward airway hyperrespon-siveness (AHR) and the development of Th2-type CD4(+) T cells. Tim-1 expressed by Th2 CD4(+) T cells has been proposed to function as a co-stimulatory molecule. Tim-1 is also expressed by B cells, macrophages, and dendritic cells, but its role in responses by these cell types has not been firmly established. Here, we generated Tim-1-deficient mice to determine the role of Tim-1 in a murine model of allergic airway disease that depends on the development and function of Th2 effector cells and results in the generation of AHR. We found antigen-driven recruitment of inflammatory cells into airways is increased in Tim-1-deficient mice relative to WT mice. In addition, we observed increased antigen-specific cytokine production by splenocytes from antigen-sensitized Tim-1-deficient mice relative to those from controls. These data support the conclusion that Tim-1 functions in pathways that suppress recruitment of inflammatory cells into the airways and the generation or activity of CD4(+) T cells.

NFIL3/E4BP4 is a key transcription factor for CD8α⁺ dendritic cell development.

Antigen presentation by mature dendritic cells (DCs) is the first step for initiating adaptive immune responses. DCs are composed of heterogeneous functional subsets; however, the molecular mechanisms that regulate differentiation of specific DC subsets are not understood. Here, we report that the basic leucine zipper transcription factor NFIL3/E4BP4 is essential for the development of CD8α(+) conventional DCs (cDCs). Nfil3(-/-) mice specifically lack CD8α(+) cDCs but not CD8α(-) cDCs or plasmacytoid DCs in lymphoid tissues. Flt3 ligand-dependent generation of CD8α(+) cDCs in lymphoid tissues and CD8α(+)-equivalent cDCs from Nfil3(-/-) bone marrow cells was also impaired. NFIL3 regulates CD8α(+) cDC development in part through Batf3 expression. Importantly, Nfil3(-/-) mice exhibited impaired cross-priming of CD8(+) T cells against cell-associated antigen, a process normally performed by CD8α(+) cDCs, and failed to produce IL-12 after TLR3 stimulation. Thus, NFIL3 plays an essential role in the development of CD8α(+) cDCs.

NFIL3 is a regulator of IL-12 p40 in macrophages and mucosal immunity.

Regulation of innate inflammatory responses against the enteric microbiota is essential for the maintenance of intestinal homeostasis. Key participants in innate defenses are macrophages. In these studies, the basic leucine zipper protein, NFIL3, is identified as a regulatory transcription factor in macrophages, controlling IL-12 p40 production induced by bacterial products and the enteric microbiota. Exposure to commensal bacteria and bacterial products induced NFIL3 in cultured macrophages and in vivo. The Il12b promoter has a putative DNA-binding element for NFIL3. Basal and LPS-activated NFIL3 binding to this site was confirmed by chromatin immunoprecipitation. LPS-induced Il12b promoter activity was inhibited by NFIL3 expression and augmented by NFIL3-short hairpin RNA in an Il12b-bacterial artificial chromosome-GFP reporter macrophage line. Il12b inhibition by NFIL3 does not require IL-10 expression, but a C-terminal minimal repression domain is necessary. Furthermore, colonic CD11b(+) lamina propria mononuclear cells from Nfil3(-/-) mice spontaneously expressed Il12b mRNA. Importantly, lower expression of NFIL3 was observed in CD14(+) lamina propria mononuclear cells from Crohn's disease and ulcerative colitis patients compared with control subjects. Likewise, no induction of Nfil3 was observed in colons of colitis-prone Il10(-/-) mice transitioned from germ-free to a conventional microbiota. In conclusion, these experiments characterize NFIL3 as an Il12b transcriptional inhibitor. Interactions of macrophages with the enteric microbiota induce NFIL3 to limit their inflammatory capacity. Furthermore, altered intestinal NFIL3 expression may have implications for the pathogenesis of experimental and human inflammatory bowel diseases.

A SOCS-1 promoter variant is associated with total serum IgE levels.

SOCS-1 is a critical regulator of multiple signaling pathways, including those activated by cytokines that regulate Ig H chain class switching to IgE. Analysis of mice with mutations in the SOCS-1 gene demonstrated that IgE levels increase with loss of SOCS-1 alleles. This suggested that overall SOCS-1 acts as an inhibitor of IgE expression in vivo. A genetic association study was performed in 474 children enrolled in the Tucson Children's Respiratory Study to determine if genetic variation in the SOCS-1 locus correlates with altered levels of IgE. Carriers of the C-allele for a novel, 3' genomic single nucleotide polymorphism (SNP) in the SOCS-1 gene (SOCS1+1125G > C; rs33932899) were found to have significantly lower levels of serum IgE compared with those of homozygotes for the G-allele. Analysis demonstrated that the SOCS1+1125G > C SNP was in complete linkage disequilibrium with an SNP at position SOCS1-820G > T (rs33977706) of the SOCS-1 promoter. Carriers of the T-allele at the SOCS1-820G > T were also found to be associated with the decreased IgE. The promoter SNP increased transcriptional activity of the SOCS-1 promoter in reporter assays and human B cells. Consistent with this observation, the presence of this polymorphism within the promoter abolished binding of yin yang-1, which is identified as a negative regulator of SOCS-1 transcriptional activity. These data suggest that genetic variation in the SOCS-1 promoter may affect IgE production.

IL-4-induced transcription factor NFIL3/E4BP4 controls IgE class switching.

IL-4 signaling promotes IgE class switching through STAT6 activation and the induction of Ig germ-line epsilon (GLepsilon) transcription. Previously, we and others identified a transcription factor, Nfil3, as a gene induced by IL-4 stimulation in B cells. However, the precise roles of nuclear factor, IL-3-regulated (NFIL3) in IL-4 signaling are unknown. Here, we report that NFIL3 is important for IgE class switching. NFIL3-deficient mice show impaired IgE class switching, and this defect is B-cell intrinsic. The induction of GLepsilon transcripts after LPS and IL-4 stimulation is significantly reduced in NFIL3-deficient B cells. Expression of NFIL3 in NFIL3-deficient B cells restores the impairment of IgE production, and overexpression of NFIL3 in the presence of cycloheximide induces GLepsilon transcripts. Moreover, NFIL3 binds to Iepsilon promoter in vivo. Together, these results identify NFIL3 as a key regulator of IL-4-induced GLepsilon transcription in response to IL-4 and subsequent IgE class switching.

The developmental regulator protein Gon4l associates with protein YY1, co-repressor Sin3a, and histone deacetylase 1 and mediates transcriptional repression.

Genetic studies involving zebrafish and mice have demonstrated that the protein Gon4l (Gon4-like) is essential for hematopoiesis. These studies also suggested that Gon4l regulates gene expression during hematopoietic development, yet the biochemical function of Gon4l has not been defined. Here, we describe the identification of factors that interact with Gon4l and may cooperate with this protein to regulate gene expression. As predicted by polypeptide sequence conservation, Gon4l interacted and co-localized with the DNA-binding protein YY1 (Yin Yang 1). Density gradient sedimentation analysis of protein lysates from mouse M12 B cells showed that Gon4l and YY1 co-sediment with the transcriptional co-repressor Sin3a and its functional partner histone deacetylase (HDAC) 1. Consistent with these results, immunoprecipitation studies showed that Gon4l associates with Sin3a, HDAC1, and YY1 as a part of complexes that form in M12 cells. Sequential immunoprecipitation studies demonstrated that Gon4l, YY1, Sin3a, and HDAC1 could all associate as components of a single complex and that a conserved domain spanning the central portion of Gon4l was required for formation of this complex. When targeted to DNA, Gon4l repressed the activity of a nearby promoter, which correlated with the ability to interact with Sin3a and HDAC1. Our data suggest that Sin3a, HDAC1, and YY1 are co-factors for Gon4l and that Gon4l may function as a platform for the assembly of complexes that regulate gene expression.

The Nalp3 inflammasome is essential for the development of silicosis.

Inhalation of crystalline silica and asbestos is known to cause the progressive pulmonary fibrotic disorders silicosis and asbestosis, respectively. Although alveolar macrophages are believed to initiate these inflammatory responses, the mechanism by which this occurs has been unclear. Here we show that the inflammatory response and subsequent development of pulmonary fibrosis after inhalation of silica is dependent on the Nalp3 inflammasome. Stimulation of macrophages with silica results in the activation of caspase-1 in a Nalp3-dependent manner. Macrophages deficient in components of the Nalp3 inflammasome were incapable of secreting the proinflammatory cytokines interleukin (IL)-1beta and IL-18 in response to silica. Similarly, asbestos was capable of activating caspase-1 in a Nalp3-dependent manner. Activation of the Nalp3 inflammasome by silica required both an efflux of intracellular potassium and the generation of reactive oxygen species. This study demonstrates a key role for the Nalp3 inflammasome in the pathogenesis of pneumoconiosis.

Critical role of Syk-dependent STAT1 activation in innate antiviral immunity.

The JAK/STAT1 pathway is generally activated by cytokines, providing essential antiviral defense. Here, we identify that STAT1 activation is independent of cytokines and JAKs at the early infection stage of some viruses, including influenza A virus (IAV). Instead, STAT1 is activated mainly through spleen tyrosine kinase (Syk) downstream of retinoic acid-inducible gene-I/mitochondrial antiviral-signaling protein (RIG-I/MAVS) signaling. Syk deletion profoundly impairs immediate innate immunity, as evidenced by the finding that Syk deletion attenuates tyrosine phosphorylation of STAT1 and reduces the expressions of interferon-stimulated genes (ISGs) in vitro and in vivo. The antiviral response to IAV infection is also significantly suppressed in the STAT1Y701F knockin mice. The results demonstrate that STAT1 activation is dependent on Syk rather than the cytokine-activated JAK signaling at the early stage of viral infection, which is critical for initial antiviral immunity. Our finding provides insights into the complicated mechanisms underlying host immune responses to viral infection.

Antigen-specific immune responses to influenza vaccine in utero.

Initial immune responses to allergens may occur before birth, thereby modulating the subsequent development of atopy. This paradigm remains controversial, however, due to the inability to identify antigen-specific T cells in cord blood. The advent of MHC tetramers has revolutionized the detection of antigen-specific T cells. Tetramer staining of cord blood after CMV infection has demonstrated that effective CD8(+) antigen-specific immune responses can follow intrauterine viral infections. We hypothesized that sensitization to antigens occurs in utero in humans. We studied cord blood B and T cell immune responses following vaccination against influenza during pregnancy. Anti-Fluzone and anti-matrix protein IgM antibodies were detected in 38.5% (27 of 70) and 40.0% (28 of 70), respectively, of cord blood specimens. Using MHC tetramers, HA-specific CD4(+) T cells were detected among 25.0% (3 of 12) and 42.9% (6 of 14) of cord blood specimens possessing DRB1*0101 and DRB1*0401 HLA types, respectively, and were detected even when the DRB1 HLA type was inherited from the father. Matrix protein-specific CD8(+) T cells were detected among 10.0% (2 of 20) of HLA-A*0201(+) newborns. These results suggest that B and T cell immune responses occur in the fetus following vaccination against influenza and have important implications for determining when immune responses to environmental exposures begin.

The transcriptional regulator NFIL3 controls IgE production.

Cytokines are essential modulators of the immune response that underlies the inflammatory component of atopic asthma and other allergic diseases, lnterleukin-4 is an important cytokine for the regulation of allergic immune responses. However, the molecular mechanisms that regulate the response of cells to IL-4 are still not completely defined. IL-4 plays an important role in B cell biology. It can regulate B cell differentiation. For example, IL-4 induces immunoglobulin heavy chain class switching to IgE by inducing germline immunoglobulin heavy chain transcription. It also induces expression of CD23 and MHC class II. Further understanding of the mechanisms by which IL-4 mediates these biologic responses may lead to novel mechanisms for therapeutic intervention and control of allergy. To define how different signaling pathways activated by IL-4 regulate gene transcription, we identified many differentially expressed genes by IL-4 stimulation by microarray analysis. NFIL3 (nuclear factor, interleukin 3 regulated) is the most strongly induced transcription factor by IL-4 stimulation in a STAT6-dependent manner. To analyze the role of NFIL3 in the immune system, we have generated NFIL3-deficient mice. NFIL3-deficient mice showed greatly impaired IgE production in response to antigen. NFIL3-deficient B cells fail to produce IgE in response to LPS plus IL-4. These defects may be due to the reduced production of immunoglobulin heavy chain germline epsilon transcripts in the absence of NFIL3. Moreover, NFIL3 KO mice sensitized and challenged with ovalbumin showed reduced airway hyper-responsiveness when compared to wild-type mice. Therefore, we hypothesize that NFIL3 is a critical regulator for IgE production and airway hyper-responsiveness.

Loss of SOCS7 in mice results in severe cutaneous disease and increased mast cell activation.

The Suppressor of Cytokine Signaling (SOCS) protein family plays a central role in the negative regulation of cytokine action and has been implicated in the development of atopic diseases. Lack of SOCS7 is associated with severe skin disease in mice. We sought to explore the underlying mechanisms resulting in this phenotype. Skin samples were analyzed and serum immunoglobulin production was measured. Cytokine production by bone marrow derived mast cells was determined by ELISA. Mast cell thymic stromal lymphopoietin (TSLP) production was assessed by quantitative real-time PCR. Data obtained revealed that Socs7(-/-) mice have increased serum IgE and IgG(1) production and exhibit an increased mast cell infiltrate, as well as un-provoked mast cell degranulation in the dermis as compared to controls. In vitro, bone marrow derived mast cells from Socs7(-/-) mice are hyperactive to IgE-mediated stimuli, with elevated production of pro-inflammatory cytokines (IL-13, IL-6, TNF-alpha). Further, activated Socs7(-/-) bone marrow derived mast cells have increased IL-7Ralpha transcript, which is part of the heterodimeric receptor for TSLP. Finally, lack of SOCS7 was accompanied by an increase in TSLP mRNA and protein production by mast cells following FcepsilonRI aggregation. These data implicate SOCS7 in the modulation of allergic inflammation and demonstrate that SOCS7 is involved in the regulation of TSLP signaling in mast cells.

Deletion of SOCS7 leads to enhanced insulin action and enlarged islets of Langerhans.

NIDDM is characterized by progressive insulin resistance and the failure of insulin-producing pancreatic beta cells to compensate for this resistance. Hyperinsulinemia, inflammation, and prolonged activation of the insulin receptor (INSR) have been shown to induce insulin resistance by decreasing INSR substrate (IRS) protein levels. Here we describe a role for SOCS7 in regulating insulin signaling. Socs7-deficient mice exhibited lower glucose levels and prolonged hypoglycemia during an insulin tolerance test and increased glucose clearance in a glucose tolerance test. Six-month-old Socs7-deficient mice exhibited increased growth of pancreatic islets with mildly increased fasting insulin levels and hypoglycemia. These defects correlated with increased IRS protein levels and enhanced insulin action in cells lacking SOCS7. Additionally, SOCS7 associated with the INSR and IRS1--molecules that are essential for normal regulation of insulin action. These data suggest that SOCS7 is a potent regulator of glucose homeostasis and insulin signaling.