The RNA helicase DDX3X is an essential mediator of innate antimicrobial immunity.

DExD/H box RNA helicases, such as the RIG-I-like receptors (RLR), are important components of the innate immune system. Here we demonstrate a pivotal and sex-specific role for the heterosomal isoforms of the DEAD box RNA helicase DDX3 in the immune system. Mice lacking DDX3X during hematopoiesis showed an altered leukocyte composition in bone marrow and spleen and a striking inability to combat infection with Listeria monocytogenes. Alterations in innate immune responses resulted from decreased effector cell availability and function as well as a sex-dependent impairment of cytokine synthesis. Thus, our data provide further in vivo evidence for an essential contribution of a non-RLR DExD/H RNA helicase to innate immunity and suggest it may contribute to sex-related differences in resistance to microbes and resilience to inflammatory disease.

ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation.

Malnutrition affects up to one billion people in the world and is a major cause of mortality. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure, cardiovascular functions and SARS infections. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea.

Localized DNA demethylation at recombination intermediates during immunoglobulin heavy chain gene assembly.

Multiple epigenetic marks have been proposed to contribute to the regulation of antigen receptor gene assembly via V(D)J recombination. Here we provide a comprehensive view of DNA methylation at the immunoglobulin heavy chain (IgH) gene locus prior to and during V(D)J recombination. DNA methylation did not correlate with the histone modification state on unrearranged alleles, indicating that these epigenetic marks were regulated independently. Instead, pockets of tissue-specific demethylation were restricted to DNase I hypersensitive sites within this locus. Though unrearranged diversity (D(H)) and joining (J(H)) gene segments were methylated, DJ(H) junctions created after the first recombination step were largely demethylated in pro-, pre-, and mature B cells. Junctional demethylation was highly localized, B-lineage-specific, and required an intact tissue-specific enhancer, Eµ. We propose that demethylation occurs after the first recombination step and may mark the junction for secondary recombination.

Development and function of murine B cells lacking RANK.

RANKL-RANK signaling regulates numerous physiologic processes such as bone remodeling, lymph node organogenesis, central thermoregulation, and formation of a lactating mammary gland in pregnancy. Recently, a receptor activator of NF-κB ligand (RANKL)-blocking Ab has been approved for human use in potentially millions of osteoporosis and cancer patients. However, germline deficiencies in RANKL or receptor activator of NF-κB (RANK) also lead to strong B cell defects in mice and human patients, suggesting that RANKL-RANK inhibition could interfere with B cell physiology and thereby trigger immunologic side-effects. To address this key question--that is, whether RANKL-RANK signaling affects B cell physiology directly or the observed defects are secondary because of the severe osteopetrosis--we generated B cell-specific RANK knockout mice. We show that B cells deficient for RANK undergo normal development and do not show any obvious defects in Ab secretion, class switch recombination, or somatic hypermutation. Our data indicate that ablation of the RANKL-RANK pathway has no direct adverse effect on B cell physiology.

Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination.

Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5' to 3', several hundred V(H) gene segments, 13 D segments, and 4 J(H) segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J(H) rearrangement occurring on both alleles before appendage of a V(H) segment. Also, IgH V(H) to DJ(H) rearrangement does not occur in T cells, even though DJ(H) rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V(H) and D clusters (generating ΔV(H)-D alleles). In both B and T cells, ΔV(H)-D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V(H) segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J(H) rearrangement and substantial V(H) to DJ(H) rearrangement of proximal IgH V(H) segments. Thus, the V(H)-D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination.

Pseudorabies virus hijacks DDX3X, initiating an addictive "mad itch" and immune suppression, to facilitate viral spread.

Infections with defined Herpesviruses, such as Pseudorabies virus (PRV) and Varicella zoster virus (VZV) can cause neuropathic itch, referred to as "mad itch" in multiple species. The underlying mechanisms involved in neuropathic "mad itch" are poorly understood. Here, we show that PRV infections hijack the RNA helicase DDX3X in sensory neurons to facilitate anterograde transport of the virus along axons. PRV induces re-localization of DDX3X from the cell body to the axons which ultimately leads to death of the infected sensory neurons. Inducible genetic ablation of Ddx3x in sensory neurons results in neuronal death and "mad itch" in mice. This neuropathic "mad itch" is propagated through activation of the opioid system making the animals "addicted to itch". Moreover, we show that PRV co-opts and diverts T cell development in the thymus via a sensory neuron-IL-6-hypothalamus-corticosterone stress pathway. Our data reveal how PRV, through regulation of DDX3X in sensory neurons, travels along axons and triggers neuropathic itch and immune deviations to initiate pathophysiological programs which facilitate its spread to enhance infectivity.

Antisense transcripts from immunoglobulin heavy-chain locus V(D)J and switch regions.

Activation-induced cytosine deaminase (AID) is essential for both somatic hypermutation (SHM) and class switch recombination (CSR), two processes involved in antibody diversification. Previously, various groups showed both in vitro and in vivo that AID initiates SHM and CSR by deaminating cytosines in DNA in a transcription-dependent manner. Although in vivo both DNA strands are equally targeted by AID, many in vitro and bacterial experiments found that AID almost exclusively targets the nontemplate strand of a transcribed substrate. Here, we report the detection of antisense transcripts in assembled Ig heavy chain (IgH) variable region exons and their immediate downstream region, as well as in switch regions, sequences that, respectively, are targets for SHM and CSR in vivo. In contrast, we did not detect antisense transcripts from the Cmu constant region exons, which lie between the IgH variable region exons and downstream S regions and which are not normally an AID target. Expression of the antisense variable region/flanking region and the S-region transcripts were found in all lymphocytes that transcribe these sequences in the sense direction. Steady-state levels of antisense transcripts appeared very low, and start sites potentially appeared heterogeneous. We discuss the potential implications of antisense IgH locus transcription for AID targeting or other processes.

Cis-regulatory elements and epigenetic changes control genomic rearrangements of the IgH locus.

Immunoglobulin variable region exons are assembled from discontinuous variable (V), diversity (D), and joining (J) segments by the process of V(D)J recombination. V(D)J rearrangements of the immunoglobulin heavy chain (IgH) locus are tightly controlled in a tissue-specific, ordered and allele-specific manner by regulating accessibility of V, D, and J segments to the recombination activating gene proteins which are the specific components of the V(D)J recombinase. In this review we discuss recent advances and established models brought forward to explain the mechanisms underlying accessibility control of V(D)J recombination, including research on germline transcripts, spatial organization, and chromatin modifications of the immunoglobulin heavy chain (IgH) locus. Furthermore, we review the functions of well-described and potential new cis-regulatory elements with regard to processes such as V(D)J recombination, allelic exclusion, and IgH class switch recombination.

Evolution of the immunoglobulin heavy chain class switch recombination mechanism.

To mount an optimum immune response, mature B lymphocytes can change the class of expressed antibody from IgM to IgG, IgA, or IgE through a recombination/deletion process termed immunoglobulin heavy chain (IgH) class switch recombination (CSR). CSR requires the activation-induced cytidine deaminase (AID), which has been shown to employ single-stranded DNA as a substrate in vitro. IgH CSR occurs within and requires large, repetitive sequences, termed S regions, which are parts of germ line transcription units (termed "C(H) genes") that are composed of promoters, S regions, and individual IgH constant region exons. CSR requires and is directed by germ line transcription of participating C(H) genes prior to CSR. AID deamination of cytidines in S regions appears to lead to S region double-stranded breaks (DSBs) required to initiate CSR. Joining of two broken S regions to complete CSR exploits the activities of general DNA DSB repair mechanisms. In this chapter, we discuss our current knowledge of the function of S regions, germ line transcription, AID, and DNA repair in CSR. We present a model for CSR in which transcription through S regions provides DNA substrates on which AID can generate DSB-inducing lesions. We also discuss how phosphorylation of AID may mediate interactions with cofactors that facilitate access to transcribed S regions during CSR and transcribed variable regions during the related process of somatic hypermutation (SHM). Finally, in the context of this CSR model, we further discuss current findings that suggest synapsis and joining of S region DSBs during CSR have evolved to exploit general mechanisms that function to join widely separated chromosomal DSBs.

The Tumor Suppressor Hace1 Is a Critical Regulator of TNFR1-Mediated Cell Fate.

The HECT domain E3 ligase HACE1 has been identified as a tumor suppressor in multiple cancers. Here, we report that HACE1 is a central gatekeeper of TNFR1-induced cell fate. Genetic inactivation of HACE1 inhibits TNF-stimulated NF-κB activation and TNFR1-NF-κB-dependent pathogen clearance in vivo. Moreover, TNF-induced apoptosis was impaired in hace1 mutant cells and knockout mice in vivo. Mechanistically, HACE1 is essential for the ubiquitylation of the adaptor protein TRAF2 and formation of the apoptotic caspase-8 effector complex. Intriguingly, loss of HACE1 does not impair TNFR1-mediated necroptotic cell fate via RIP1 and RIP3 kinases. Loss of HACE1 predisposes animals to colonic inflammation and carcinogenesis in vivo, which is markedly alleviated by genetic inactivation of RIP3 kinase and TNFR1. Thus, HACE1 controls TNF-elicited cell fate decisions and exerts tumor suppressor and anti-inflammatory activities via a TNFR1-RIP3 kinase-necroptosis pathway.

Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense.

Neutrophils are key innate immune effector cells that are essential to fighting bacterial and fungal pathogens. Here we report that mice carrying a hematopoietic lineage-specific deletion of Jagn1 (encoding Jagunal homolog 1) cannot mount an efficient neutrophil-dependent immune response to the human fungal pathogen Candida albicans. Global glycobiome analysis identified marked alterations in the glycosylation of proteins involved in cell adhesion and cytotoxicity in Jagn1-deficient neutrophils. Functional analysis confirmed marked defects in neutrophil migration in response to Candida albicans infection and impaired formation of cytotoxic granules, as well as defective myeloperoxidase release and killing of Candida albicans. Treatment with granulocyte/macrophage colony-stimulating factor (GM-CSF) protected mutant mice from increased weight loss and accelerated mortality after Candida albicans challenge. Notably, GM-CSF also restored the defective fungicidal activity of bone marrow cells from humans with JAGN1 mutations. These data directly identify Jagn1 (JAGN1 in humans) as a new regulator of neutrophil function in microbial pathogenesis and uncover a potential treatment option for humans.

A dual role for autophagy in a murine model of lung cancer.

Autophagy is a mechanism by which starving cells can control their energy requirements and metabolic states, thus facilitating the survival of cells in stressful environments, in particular in the pathogenesis of cancer. Here we report that tissue-specific inactivation of Atg5, essential for the formation of autophagosomes, markedly impairs the progression of KRas(G12D)-driven lung cancer, resulting in a significant survival advantage of tumour-bearing mice. Autophagy-defective lung cancers exhibit impaired mitochondrial energy homoeostasis, oxidative stress and a constitutively active DNA damage response. Genetic deletion of the tumour suppressor p53 reinstates cancer progression of autophagy-deficient tumours. Although there is improved survival, the onset of Atg5-mutant KRas(G12D)-driven lung tumours is markedly accelerated. Mechanistically, increased oncogenesis maps to regulatory T cells. These results demonstrate that, in KRas(G12D)-driven lung cancer, Atg5-regulated autophagy accelerates tumour progression; however, autophagy also represses early oncogenesis, suggesting a link between deregulated autophagy and regulatory T cell controlled anticancer immunity.

ACE2 - from the renin-angiotensin system to gut microbiota and malnutrition.

The renin-angiotensin system (RAS) is a complex network that regulates blood pressure, electrolyte and fluid homeostasis, as well as the function of several organs. Angiotensin-converting enzyme 2 (ACE2) was identified as an enzyme that negatively regulates the RAS by converting Ang II, the main bioactive molecule of the RAS, to Ang 1-7. Thus, ACE2 counteracts the role of angiotensin-converting enzyme (ACE) which generates Ang II from Ang I. ACE and ACE2 have been implicated in several pathologies such as cardiovascular and renal disease or acute lung injury. In addition, ACE2 has functions independent of the RAS: ACE2 is the receptor for the SARS coronavirus and ACE2 is essential for expression of neutral amino acid transporters in the gut. In this context, ACE2 modulates innate immunity and influences the composition of the gut microbiota, which can explain diarrhea and intestinal inflammation observed in Hartnup disorder, Pellagra, or under conditions of severe malnutrition. Here we review and discuss the diverse functions of ACE2 and its relevance to human pathologies.

Analysis of mice lacking DNaseI hypersensitive sites at the 5' end of the IgH locus.

The 5' end of the IgH locus contains a cluster of DNaseI hypersensitive sites, one of which (HS1) was shown to be pro-B cell specific and to contain binding sites for the transcription factors PU.1, E2A, and Pax5. These data as well as the location of the hypersensitive sites at the 5' border of the IgH locus suggested a possible regulatory function for these elements with respect to the IgH locus. To test this notion, we generated mice carrying targeted deletions of either the pro-B cell specific site HS1 or the whole cluster of DNaseI hypersensitive sites. Lymphocytes carrying these deletions appear to undergo normal development, and mutant B cells do not exhibit any obvious defects in V(D)J recombination, allelic exclusion, or class switch recombination. We conclude that deletion of these DNaseI hypersensitive sites does not have an obvious impact on the IgH locus or B cell development.

A 220-nucleotide deletion of the intronic enhancer reveals an epigenetic hierarchy in immunoglobulin heavy chain locus activation.

A tissue-specific transcriptional enhancer, Emu, has been implicated in developmentally regulated recombination and transcription of the immunoglobulin heavy chain (IgH) gene locus. We demonstrate that deleting 220 nucleotides that constitute the core Emu results in partially active locus, characterized by reduced histone acetylation, chromatin remodeling, transcription, and recombination, whereas other hallmarks of tissue-specific locus activation, such as loss of H3K9 dimethylation or gain of H3K4 dimethylation, are less affected. These observations define Emu-independent and Emu-dependent phases of locus activation that reveal an unappreciated epigenetic hierarchy in tissue-specific gene expression.

S-S synapsis during class switch recombination is promoted by distantly located transcriptional elements and activation-induced deaminase.

Molecular mechanisms underlying synapsis of activation-induced deaminase (AID)-targeted S regions during class switch recombination (CSR) are poorly understood. By using chromosome conformation capture techniques, we found that in B cells, the Emicro and 3'Ealpha enhancers were in close spatial proximity, forming a unique chromosomal loop configuration. B cell activation led to recruitment of the germline transcript (GLT) promoters to the Emicro:3'Ealpha complex in a cytokine-dependent fashion. This structure facilitated S-S synapsis because Smicro was proximal to Emicro and a downstream S region was corecruited with the targeted GLT promoter to Emicro:3'Ealpha. We propose that GLT promoter association with the Emicro:3'Ealpha complex creates an architectural scaffolding that promotes S-S synapsis during CSR and that these interactions are stabilized by AID. Thus, the S-S synaptosome is formed as a result of the self-organizing transcription system that regulates GLT expression and may serve to guard against spurious chromosomal translocations.

Gene repression by Pax5 in B cells is essential for blood cell homeostasis and is reversed in plasma cells.

The transcription factor Pax5 represses lineage-inappropriate genes and activates B cell-specific genes in B lymphocytes. By identifying 110 Pax5-repressed genes, we now demonstrate that Pax5 downregulates diverse biological activities including receptor signaling, cell adhesion, migration, transcriptional control, and cellular metabolism at B cell commitment. The T lymphoid or myeloid expression of these genes demonstrates that Pax5(-/-) pro-B cells and common lymphoid progenitors display lymphoid and myeloid promiscuity of gene expression. These lineage-inappropriate genes require continuous Pax5 activity for their repression, as they are reactivated in committed pro-B cells and mature B cells following conditional Pax5 deletion. Pax5-repressed genes are also reexpressed in plasma cells, which depend for normal function on Cd28 and Ccr2 reactivation. The loss of Pax5 during terminal differentiation thus contributes to the plasma cell transcription program. Finally, ectopic expression of the Pax5-repressed chemokine gene Ccl3 in B cells results in increased osteoclast formation and bone loss, demonstrating that Pax5-mediated gene repression is essential for normal homeostasis of hematopoietic development.

Elucidation of IgH intronic enhancer functions via germ-line deletion.

Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iEmu) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iEmu (cEmu) were either deleted (cEmu(Delta/Delta) mice) or replaced with a pgk-Neo(R) cassette (cEmu(N/N) mice). The cEmu(Delta/Delta) mice had reduced B cell numbers, in association with impaired D to J(H) and V(H) to DJ(H) rearrangement, whereas cEmu(N/N) mice had a complete block in IgH V(D)J(H) recombination, confirming that additional cis elements cooperate with iEmu to enforce D to J(H) recombination. In addition, developing cEmu(Delta/Delta) and cEmu(N/N) B lineage cells had correspondingly decreased levels of germ-line transcripts from the J(H) region of the IgH locus (mu0 and Imu transcripts); although both had normal levels of germ-line V(H) transcripts, suggesting that cEmu may influence IgH locus V(D)J recombination by influencing accessibility of J(H) proximal regions of the locus. Consistent with chimera studies, peripheral cEmu(Delta/Delta) B cells had normal surface Ig and relatively normal class switch recombination. However, cEmu(Delta/Delta) B cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cEmu is not required for this process. The availability of mice with the iEmu mutation in their germ line will facilitate future studies to elucidate the roles of iEmu in V(H)(D)J(H) recombination in the context of IgH chromatin structure and germ-line transcription.

ACE2 links amino acid malnutrition to microbial ecology and intestinal inflammation

Malnutrition affects up to one billion people in the world and is a major cause of mortality. In many cases, malnutrition is associated with diarrhoea and intestinal inflammation, further contributing to morbidity and death. The mechanisms by which unbalanced dietary nutrients affect intestinal homeostasis are largely unknown. Here we report that deficiency in murine angiotensin I converting enzyme (peptidyl-dipeptidase A) 2 (Ace2), which encodes a key regulatory enzyme of the renin-angiotensin system (RAS), results in highly increased susceptibility to intestinal inflammation induced by epithelial damage. The RAS is known to be involved in acute lung failure, cardiovascular functions and SARS infections. Mechanistically, ACE2 has a RAS-independent function, regulating intestinal amino acid homeostasis, expression of antimicrobial peptides, and the ecology of the gut microbiome. Transplantation of the altered microbiota from Ace2 mutant mice into germ-free wild-type hosts was able to transmit the increased propensity to develop severe colitis. ACE2-dependent changes in epithelial immunity and the gut microbiota can be directly regulated by the dietary amino acid tryptophan. Our results identify ACE2 as a key regulator of dietary amino acid homeostasis, innate immunity, gut microbial ecology, and transmissible susceptibility to colitis. These results provide a molecular explanation for how amino acid malnutrition can cause intestinal inflammation and diarrhoea(AU)