B cell expression of E3 ubiquitin ligase Cul4b promotes chronic gammaherpesvirus infection in vivo.

IMPORTANCE: The human gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus are etiologic agents of numerous B cell lymphomas. A hallmark of gammaherpesvirus infection is their ability to establish lifelong latency in B cells. However, the specific mechanisms that mediate chronic infection in B cells in vivo remain elusive. Cellular E3 ubiquitin ligases regulate numerous biological processes by catalyzing ubiquitylation and modifying protein location, function, or half-life. Many viruses hijack host ubiquitin ligases to evade antiviral host defense and promote viral fitness. Here, we used the murine gammaherpesvirus 68 in vivo system to demonstrate that the E3 ligase Cul4b is essential for this virus to establish latency in germinal center B cells. These findings highlight an essential role for this E3 ligase in promoting chronic gammaherpesvirus infection in vivo and suggest that targeted inhibition of E3 ligases may provide a novel and effective intervention strategy against gammaherpesvirus-associated diseases.

Gammaherpesvirus-mediated repression reveals EWSR1 to be a negative regulator of B cell responses.

The germinal center (GC) plays a central role in the generation of antigen-specific B cells and antibodies. Tight regulation of the GC is essential due to the inherent risks of tumorigenesis and autoimmunity posed by inappropriate GC B cell processes. Gammaherpesviruses such as Epstein-Barr virus (EBV) and murine gammaherpesvirus 68 (MHV68) utilize numerous armaments to drive infected naïve B cells, independent of antigen, through GC reactions to expand the latently infected B cell population and establish a stable latency reservoir. We previously demonstrated that the MHV68 microRNA (miRNA) mghv-miR-M1-7-5p represses host EWSR1 (Ewing sarcoma breakpoint region 1) to promote B cell infection. EWSR1 is a transcription and splicing regulator that is recognized for its involvement as a fusion protein in Ewing sarcoma. A function for EWSR1 in B cell responses has not been previously reported. Here, we demonstrate that 1) B cell-specific deletion of EWSR1 had no effect on generation of mature B cell subsets or basal immunoglobulin levels in naïve mice, 2) repression or ablation of EWSR1 in B cells promoted expansion of MHV68 latently infected GC B cells, and 3) B cell-specific deletion of EWSR1 during a normal immune response to nonviral antigen resulted in significantly elevated numbers of antigen-specific GC B cells, plasma cells, and circulating antibodies. Notably, EWSR1 deficiency did not affect the proliferation or survival of GC B cells but instead resulted in the generation of increased numbers of precursor GC B cells. Cumulatively, these findings demonstrate that EWSR1 is a negative regulator of B cell responses.

Metabolic regulation of follicular helper T cell differentiation in a mouse model of lupus.

Follicular helper T (TFH) cells are expanded in systemic lupus erythematosus (SLE), where they are required for production of high affinity autoantibodies. A better understanding of the mechanisms that regulate the differentiation of TFH cells is critical. Naïve T cells from lupus-prone B6.NZM2410.Sle1.Sle2.Sle3 (TC) mice showed an intrinsic higher capacity to differentiate into TFH cells. Metabolic reprogramming is a vital regulatory mechanism for T cell differentiation, but how metabolic pathways contribute to TFH cell expansion in SLE remains elusive. Here we show that glycolysis, mTOR signaling, FAO, and the activity of complex V of the electron transport chain support TFH lineage commitment. Blocking complex I uniquely decreased the expansion of TFH cells from lupus-prone mice, and inhibition of some pathways had a greater effect in lupus-prone than control TFH cells. However, blocking glutaminolysis, complex III and ADP/ATP translocase did not affect TFH cell expansion. Together, our results identified novel intrinsic metabolic requirements for TFH cell differentiation, and further defined the differential metabolic pathways that support the expansion of TFH cells in lupus-prone mice. Together, our data indicates the crucial but distinct roles for metabolic pathways in TFH cell differentiation and provide a comprehensive experimental basis for fully understanding the precise roles of distant metabolic signaling in regulating the TFH cell differentiation.

The ubiquitin ligase Cul5 regulates CD4+ T cell fate choice and allergic inflammation.

Antigen encounter directs CD4+ T cells to differentiate into T helper or regulatory cells. This process focuses the immune response on the invading pathogen and limits tissue damage. Mechanisms that govern T helper cell versus T regulatory cell fate remain poorly understood. Here, we show that the E3 ubiquitin ligase Cul5 determines fate selection in CD4+ T cells by regulating IL-4 receptor signaling. Mice lacking Cul5 in T cells develop Th2 and Th9 inflammation and show pathophysiological features of atopic asthma. Following T cell activation, Cul5 forms a complex with CIS and pJak1. Cul5 deletion reduces ubiquitination and subsequent degradation of pJak1, leading to an increase in pJak1 and pSTAT6 levels and reducing the threshold of IL-4 receptor signaling. As a consequence, Cul5 deficient CD4+ T cells deviate from Treg to Th9 differentiation in low IL-4 conditions. These data support the notion that Cul5 promotes a tolerogenic T cell fate choice and reduces susceptibility to allergic asthma.

Immune Dysregulation in Human ITCH Deficiency Successfully Treated with Hematopoietic Cell Transplantation.

BACKGROUND: Mutations in ITCH, which encodes an E3 ubiquitin-protein ligase, can result in systemic autoimmunity and immunodeficiency. The clinical phenotype and mechanism of disease have not been fully characterized, resulting in a paucity of therapeutic options for this potentially fatal disease. OBJECTIVE: We aimed to (1) expand the understanding about the phenotype of human ITCH deficiency (2) further characterize the associated immune dysregulation, and (3) report the first successful hematopoietic cell transplant (HCT) in a patient with ITCH deficiency. METHODS: Disease profiling was performed in a patient with multisystem immune dysregulation. Whole exome sequencing with trio analysis and functional validation of candidate disease variants were performed, including mRNA and protein expression. Analyses to further delineate the immunophenotype included quantitative evaluation of lymphoid and myeloid subsets with flow cytometry and mass cytometry. RESULTS: A patient with multisystem immune dysregulation presenting with growth failure, very-early-onset inflammatory bowel disease, arthritis, uveitis, psoriasis, and type 1 diabetes mellitus underwent whole exome sequencing, which identified novel compound heterozygous mutations in ITCH. Reduced expression of ITCH mRNA and absent ITCH protein were found. Abnormalities in both lymphoid and myeloid lineages were identified. The patient underwent HCT. He demonstrated excellent immune reconstitution and resolution of many manifestations of his systemic disease. CONCLUSIONS: Here we report ITCH deficiency with unique clinical features of colonic very-early-onset inflammatory bowel disease, arthritis, and uveitis in the setting of immune dysregulation and further characterize the underlying immune dysregulation. We demonstrate that HCT can be an effective, and potentially curative, therapy for ITCH deficiency.

The E3 ubiquitin ligase Cul4b promotes CD4+ T cell expansion by aiding the repair of damaged DNA.

The capacity for T cells to become activated and clonally expand during pathogen invasion is pivotal for protective immunity. Our understanding of how T cell receptor (TCR) signaling prepares cells for this rapid expansion remains limited. Here we provide evidence that the E3 ubiquitin ligase Cullin-4b (Cul4b) regulates this process. The abundance of total and neddylated Cul4b increased following TCR stimulation. Disruption of Cul4b resulted in impaired proliferation and survival of activated T cells. Additionally, Cul4b-deficient CD4+ T cells accumulated DNA damage. In T cells, Cul4b preferentially associated with the substrate receptor DCAF1, and Cul4b and DCAF1 were found to interact with proteins that promote the sensing or repair of damaged DNA. While Cul4b-deficient CD4+ T cells showed evidence of DNA damage sensing, downstream phosphorylation of SMC1A did not occur. These findings reveal an essential role for Cul4b in promoting the repair of damaged DNA to allow survival and expansion of activated T cells.

Targeting the ion channel TRPM7 promotes the thymic development of regulatory T cells by promoting IL-2 signaling.

The thymic development of regulatory T (Treg) cells, crucial suppressors of the responses of effector T (Teff) cells, is governed by the transcription factor FOXP3. Despite the clinical importance of Treg cells, there is a dearth of druggable molecular targets capable of increasing their numbers in vivo. We found that inhibiting the function of the TRPM7 chanzyme (ion channel and enzyme) potentiated the thymic development of Treg cells in mice and led to a substantially higher frequency of functional Treg cells in the periphery. In addition, TRPM7-deficient mice were resistant to T cell-driven hepatitis. Deletion of Trpm7 and inhibition of TRPM7 channel activity by the FDA-approved drug FTY720 increased the sensitivity of T cells to the cytokine interleukin-2 (IL-2) through a positive feed-forward loop involving increased expression of the IL-2 receptor α-subunit and activation of the transcriptional regulator STAT5. Enhanced IL-2 signaling increased the expression of Foxp3 in thymocytes and promoted thymic Treg (tTreg) cell development. Thus, these data indicate that inhibiting TRPM7 activity increases Treg cell numbers, suggesting that it may be a therapeutic target to promote immune tolerance.

Itch regulation of innate and adaptive immune responses in mice and humans.

The E3 ubiquitin ligase Itch has long been appreciated to be a critical suppressor of inflammation, first identified as a regulator of Th2 differentiation and lung inflammation. Recent studies have revealed novel roles for this protein in mouse and human disease, and it is now clear that Itch also limits the function of other lymphocytes, innate immune cells, and nonhematopoietic cells to regulate immunity. In addition to Th2 cells, Itch also regulates Th17 and regulatory T cells. Itch regulates humoral immunity through direct roles in T follicular helper cells and T follicular regulatory cells, and B cells. Furthermore, Itch limits innate immune responses, such as macrophage cytokine production. Through these cell-intrinsic functions, Itch regulates the interplay between innate and adaptive immune cells, resulting in profound autoinflammation in Itch-deficient mice. Whereas Itch deficiency was previously thought to be an extremely rare occurrence humans, whole exome sequencing of patients with unexplained autoimmune disease has revealed at least two additional cases of Itch deficiency in the last year alone, each caused by distinct mutations within the Itch gene. The recent identification of these patients suggests that Itch mutations may be more common than previously thought, and demonstrates the need to understand how this protein regulates inflammation and autoimmune disease.

Itch attenuates CD4 T-cell proliferation in mice by limiting WBP2 protein stability.

To mount an antipathogen response, CD4 T cells must undergo rapid cell proliferation; however, poorly controlled expansion can result in diseases such as autoimmunity. One important regulator of T-cell activity is the E3 ubiquitin ligase Itch. Itch deficient patients suffer from extensive autoinflammation. Similarly, Itch deficient mice exhibit inflammation characterized by high numbers of activated CD4 T cells. While the role of Itch in limiting CD4 T-cell cytokine production has been extensively studied, it is less clear whether and how Itch regulates proliferation of these cells. We determined that Itch deficient CD4 T cells are hyperproliferative in vitro and in vivo, due to increased S phase entry. Whole cell proteomics analysis of Itch deficient primary mouse CD4 T cells revealed increased abundance of the ß-catenin coactivator WW domain-binding protein 2 (WBP2). Furthermore, Itch deficient cells demonstrate increased WBP2 protein stability, and Itch and WBP2 interact in CD4 T cells. Knockdown of WBP2 in CD4 T cells caused reduced proliferation. Together, our data support that Itch attenuates CD4 T cell proliferation by promoting WBP2 degradation. This study identifies novel roles for Itch and WBP2 in regulating CD4 T cell proliferation, providing insight into how Itch may prevent inflammation.

The E3 ubiquitin ligase Itch restricts antigen-driven B cell responses.

The E3 ubiquitin ligase Itch regulates antibody levels and prevents autoimmune disease in humans and mice, yet how Itch regulates B cell fate or function is unknown. We now show that Itch directly limits B cell activity. While Itch-deficient mice displayed normal numbers of preimmune B cell populations, they showed elevated numbers of antigen-experienced B cells. Mixed bone marrow chimeras revealed that Itch acts within B cells to limit naive and, to a greater extent, germinal center (GC) B cell numbers. B cells lacking Itch exhibited increased proliferation, glycolytic capacity, and mTORC1 activation. Moreover, stimulation of these cells in vivo by WT T cells resulted in elevated numbers of GC B cells, PCs, and serum IgG. These results support a novel role for Itch in limiting B cell metabolism and proliferation to suppress antigen-driven B cell responses.

Regulation of autoimmune disease by the E3 ubiquitin ligase Itch.

Itch is a HECT type E3 ubiquitin ligase that is required to prevent the development of autoimmune disease in both mice and humans. Itch is expressed in most mammalian cell types, and, based on published data, it regulates many cellular pathways ranging from T cell differentiation to liver tumorigenesis. Since 1998, when Itch was first discovered, hundreds of publications have described mechanisms through which Itch controls various biologic activities in both immune and non-immune cells. Other studies have provided insight into how Itch catalytic activity is regulated. However, while autoimmunity is the primary clinical feature that occurs in both mice and humans lacking Itch, and Itch control of immune cell function has been well-studied, it remains unclear how Itch prevents the emergence of autoimmune disease. In this review, we explore recent discoveries that advance our understanding of how Itch regulates immune cell biology, and the extent to which these clarify how Itch prevents autoimmune disease. Additionally, we discuss how molecular regulators of Itch impact its ability to control these processes, as this may provide clues on how to therapeutically target Itch to treat patients with autoimmune disease.

Aberrant Th2 inflammation drives dysfunction of alveolar macrophages and susceptibility to bacterial pneumonia.

The ubiquitin ligase, Itch, is required to prevent autoinflammatory disease in mice and humans. Itch-deficient mice develop lethal pulmonary inflammation characterized by the production of Th2 cytokines (for example, interleukin-4 (IL-4)); however, the contribution of Itch to immune defense against respiratory pathogens has not been determined. We found that Itch-deficient mice were highly susceptible to intranasal infection with the respiratory pathogen Klebsiella pneumoniae. Infected Itch-deficient mice exhibited increased immune cell infiltration, cytokine levels and bacterial burden in the respiratory tract compared with control mice. However, numbers of resident alveolar macrophages were reduced in the lungs from Itch-deficient mice both before and after infection. High levels of Th2 cytokines in the respiratory tract correlated with deceased alveolar macrophages, and genetic ablation of IL-4 restored alveolar macrophages and host defense to K. pneumoniae in Itch-deficient mice, suggesting that loss of alveolar macrophages occurred as a consequence of Th2 inflammation. Adoptive transfer of Itch-/- CD4+ T cells into Rag-/- mice was sufficient to drive reduction in numbers of Itch-replete alveolar macrophages. Finally, we found that Stat6 signaling downstream of the IL-4 receptor directly reduced fitness of alveolar macrophages when these cells were exposed to the Itch-/- inflamed respiratory tract. These data suggest that Th2 inflammation directly impairs alveolar macrophage fitness in Itch-/- mice, and elucidate a previously unappreciated link between Th2 cells, alveolar macrophages and susceptibility to bacterial infection.

Ndfip1 restricts mTORC1 signalling and glycolysis in regulatory T cells to prevent autoinflammatory disease.

Foxp3+ T regulatory (Treg) cells suppress immune cell activation and establish normal immune homeostasis. How Treg cells maintain their identity is not completely understood. Here we show that Ndfip1, a coactivator of Nedd4-family E3 ubiquitin ligases, is required for Treg cell stability and function. Ndfip1 deletion in Treg cells results in autoinflammatory disease. Ndfip1-deficient Treg cells are highly proliferative and are more likely to lose Foxp3 expression to become IL-4-producing TH2 effector cells. Proteomic analyses indicate altered metabolic signature of Ndfip1-deficient Treg cells and metabolic profiling reveals elevated glycolysis and increased mTORC1 signalling. Ndfip1 restricts Treg cell metabolism and IL-4 production via distinct mechanisms, as IL-4 deficiency does not prevent hyperproliferation or elevated mTORC1 signalling in Ndfip1-deficient Treg cells. Thus, Ndfip1 preserves Treg lineage stability and immune homeostasis by preventing the expansion of highly proliferative and metabolically active Treg cells and by preventing pathological secretion of IL-4 from Treg cells.

Influenza A induces dysfunctional immunity and death in MeCP2-overexpressing mice.

Loss of function or overexpression of methyl-CpG-binding protein 2 (MeCP2) results in the severe neurodevelopmental disorders Rett syndrome and MeCP2 duplication syndrome, respectively. MeCP2 plays a critical role in neuronal function and the function of cells throughout the body. It has been previously demonstrated that MeCP2 regulates T cell function and macrophage response to multiple stimuli, and that immune-mediated rescue imparts significant benefit in Mecp2-null mice. Unlike Rett syndrome, MeCP2 duplication syndrome results in chronic, severe respiratory infections, which represent a significant cause of patient morbidity and mortality. Here, we demonstrate that MeCP2Tg3 mice, which overexpress MeCP2 at levels 3- to 5-fold higher than normal, are hypersensitive to influenza A/PR/8/34 infection. Prior to death, MeCP2Tg3 mice experienced a host of complications during infection, including neutrophilia, increased cytokine production, excessive corticosterone levels, defective adaptive immunity, and vascular pathology characterized by impaired perfusion and pulmonary hemorrhage. Importantly, we found that radioresistant cells are essential to infection-related death after bone marrow transplantation. In all, these results demonstrate that influenza A infection in MeCP2Tg3 mice results in pathology affecting both immune and nonhematopoietic cells, suggesting that failure to effectively respond and clear viral respiratory infection has a complex, multicompartment etiology in the context of MeCP2 overexpression.

IL-21R signaling suppresses IL-17+ gamma delta T cell responses and production of IL-17 related cytokines in the lung at steady state and after Influenza A virus infection.

Influenza A virus (IAV) infection of the respiratory tract elicits a robust immune response, which is required for efficient virus clearance but at the same time can contribute to lung damage and enhanced morbidity. IL-21 is a member of the type I cytokine family and has many different immune-modulatory functions during acute and chronic virus infections, although its role in IAV infection has not been fully evaluated. In this report we evaluated the contributions of IL-21/IL-21 receptor (IL-21R) signaling to host defense in a mouse model of primary IAV infection using IL-21R knock out (KO) mice. We found that lack of IL-21R signaling had no significant impact on virus clearance, adaptive T cell responses, or myeloid cell accumulations in the respiratory tract. However, a subset of inflammatory cytokines were elevated in the bronchoalveolar lavage fluid of IL-21R KO mice, including IL-17. Although there was only a small increase in Th17 cells in the lungs of IL-21R KO mice, we observed a dramatic increase in gamma delta (γδ) T cells capable of producing IL-17 both after IAV infection and at steady state in the respiratory tract. Finally, we found that IL-21R signaling suppressed the accumulation of IL-17+ γδ T cells in the respiratory tract intrinsically. Thus, our study reveals a previously unrecognized role of IL-21R signaling in regulating IL-17 production by γδ T cells.

Late engagement of CD86 after influenza virus clearance promotes recovery in a FoxP3+ regulatory T cell dependent manner.

Influenza A virus (IAV) infection in the respiratory tract triggers robust innate and adaptive immune responses, resulting in both virus clearance and lung inflammation and injury. After virus clearance, resolution of ongoing inflammation and tissue repair occur during a distinct recovery period. B7 family co-stimulatory molecules such as CD80 and CD86 have important roles in modulating T cell activity during the initiation and effector stages of the host response to IAV infection, but their potential role during recovery and resolution of inflammation is unknown. We found that antibody-mediated CD86 blockade in vivo after virus clearance led to a delay in recovery, characterized by increased numbers of lung neutrophils and inflammatory cytokines in airways and lung interstitium, but no change in conventional IAV-specific T cell responses. However, CD86 blockade led to decreased numbers of FoxP3+ regulatory T cells (Tregs), and adoptive transfer of Tregs into αCD86 treated mice rescued the effect of the blockade, supporting a role for Tregs in promoting recovery after virus clearance. Specific depletion of Tregs late after infection mimicked the CD86 blockade phenotype, confirming a role for Tregs during recovery after virus clearance. Furthermore, we identified neutrophils as a target of Treg suppression since neutrophil depletion in Treg-depleted mice reduced excess inflammatory cytokines in the airways. These results demonstrate that Tregs, in a CD86 dependent mechanism, contribute to the resolution of disease after IAV infection, in part by suppressing neutrophil-driven cytokine release into the airways.

Dendritic cells tolerized with adenosine A2AR agonist attenuate acute kidney injury.

DC-mediated NKT cell activation is critical in initiating the immune response following kidney ischemia/reperfusion injury (IRI), which mimics human acute kidney injury (AKI). Adenosine is an important antiinflammatory molecule in tissue inflammation, and adenosine 2A receptor (A2AR) agonists protect kidneys from IRI through their actions on leukocytes. In this study, we showed that mice with A2AR-deficient DCs are more susceptible to kidney IRI and are not protected from injury by A2AR agonists. In addition, administration of DCs treated ex vivo with an A2AR agonist protected the kidneys of WT mice from IRI by suppressing NKT production of IFN-γ and by regulating DC costimulatory molecules that are important for NKT cell activation. A2AR agonists had no effect on DC antigen presentation or on Tregs. We conclude that ex vivo A2AR-induced tolerized DCs suppress NKT cell activation in vivo and provide a unique and potent cell-based strategy to attenuate organ IRI.

CD4+ T cell help and innate-derived IL-27 induce Blimp-1-dependent IL-10 production by antiviral CTLs.

Interleukin (IL)-10 is an important regulatory cytokine that can modulate excessive immune mediated injury. Several distinct cell types have been demonstrated to produce IL-10, including most recently CD8+ cytotoxic T lymphocytes (CTLs) responding to respiratory virus infection. Here we report that CD4+ T cell help in the form of IL-2 is required for IL-10 production by CTLs, but not for the induction of CTL effector cytokines. We show that IL-2 derived from CD4+ helper T cells cooperates with innate immune cell-derived IL-27 to amplify IL-10 production by CTLs through a Blimp-1-dependent mechanism. These findings reveal a previously unrecognized pathway that coordinates signals derived from innate and helper T cells to control the production of a regulatory cytokine by CTLs during acute viral infection.