Metazoans have developed strategies to protect themselves from pathogenic attack. These preserved mechanisms constitute the immune system, composed of innate and adaptive responses. Among the two kinds, the innate immune system involves the activation of a fast response. NF-κB signaling pathways are activated during infections and lead to the expression of timely-controlled immune response genes. However, activation of NF-κB pathways can be deleterious when uncontrolled. Their regulation is necessary to prevent the development of inflammatory diseases or cancers. The similarity of the NF-κB pathways mediating immune mechanisms in insects and mammals makes Drosophila melanogaster a suitable model for studying the innate immune response and learning general mechanisms that are also relevant for humans. In this review, we summarize what is known about the dynamic regulation of the central NF-κB-pathways and go into detail on the molecular level of the IMD pathway. We report on the role of the nuclear protein Akirin in the regulation of the NF-κB Relish immune response. The use of the Drosophila model allows the understanding of the fine-tuned regulation of this central NF-κB pathway.

The Immune Deficiency (IMD) pathway in Drosophila melanogaster is activated upon microbial challenge with Gram-negative bacteria to trigger the innate immune response. In order to decipher this nuclear factor κB (NF-κB) signaling pathway, we undertook an in vitro RNAi screen targeting E3 ubiquitin ligases specifically and identified the HECT-type E3 ubiquitin ligase Hyperplastic discs (Hyd) as a new actor in the IMD pathway. Hyd mediated Lys 63 (K63)-linked polyubiquitination of the NF-κB cofactor Akirin was required for efficient binding of Akirin to the NF-κB transcription factor Relish. We showed that this Hyd-dependent interaction was required for the transcription of immunity-related genes that are activated by both Relish and Akirin but was dispensable for the transcription of genes that depend solely on Relish. Therefore Hyd is key in NF-κB transcriptional selectivity downstream of the IMD pathway. Drosophila depleted of Akirin or Hyd failed to express the full set of genes encoding immune-induced anti-microbial peptides and succumbed to immune challenges. We showed further that UBR5, the mammalian homolog of Hyd, was also required downstream of the NF-κB pathway for the activation of Interleukin 6 (IL6) transcription by LPS or IL-1β in cultured human cells. Our findings link the action of an E3 ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.

Juvenile idiopathic arthritis (JIA) is an inflammatory rheumatic disorder. Polymorphonuclear neutrophils (PMNs) are present in JIA synovial fluid (SF), but with variable frequency. SF PMNs in JIA were previously shown to display high exocytic but low phagocytic and immunoregulatory activities. To further assess whether the degree of SF neutrophilia associated with altered immune responses in JIA, we collected SF and blood from 16 adolescent JIA patients. SF and blood leukocytes were analyzed by flow cytometry. SF and plasma were used for immune mediator quantification and metabolomics. Healthy donor blood T cells were cultured in SF to evaluate its immunoregulatory activities. PMN and T cell frequencies were bimodal in JIA SF, delineating PMN high/T cell low (PMNHigh) and PMN low/T cell high (PMNLow) samples. Proinflammatory mediators were increased in SF compared with plasma across patients, and pro- and anti-inflammatory mediators were further elevated in PMNHigh SF. Compared to blood, SF PMNs showed increased exocytosis and programmed death-1/programmed death ligand-1 expression, and SF PMNs and monocytes/macrophages had increased surface-bound arginase-1. SPADE analysis revealed SF monocyte/macrophage subpopulations coexpressing programmed death-1 and programmed death ligand-1, with higher expression in PMNHigh SF. Healthy donor T cells showed reduced coreceptor expression when stimulated in PMNHigh versus PMNLow SF. However, amino acid metabolites related to the arginase-1 and IDO-1 pathways did not differ between the two groups. Hence, PMN predominance in the SF of a subset of JIA patients is associated with elevated immune mediator concentration and may alter SF monocyte/macrophage phenotype and T cell activation, without altering immunoregulatory amino acids.

Rationale Juvenile idiopathic arthritis (JIA) is an inflammatory autoimmune disorder driven by dysfunction of the joint tissue and abnormal immune responses. As deep phenotyping of synovial fluid (SF) might yield new targetable mechanisms of inflammation in JIA patients, we initiated the Emory Multi Omics JIA Immunology (EMOJI) project. Methods Patients with JIA between the ages of 6 and 18 were consented and enrolled for collection of SF from the knee and venous blood. Blood and SF leukocytes were analyzed by flow cytometry. Platelet-free plasma and debris-free SF supernatant were obtained by dual centrifugation and a 20-plex chemiluminescent assay (Meso Scale Discovery) was used to quantify cytokines. Results We report data on blood and SF collected at the same visit from 7 JIA patients. JIA subtypes represented were psoriatic (N=1), oligoarticular extended (N=1), oligoarticular persistent (N=2) and polyarticular (N=3) arthritis; all rheumatoid factor-negative. CD3 expression was decreased on SF compared to blood T-cells. Concomitantly, expression of the CD3-inhibitory enzyme arginase was increased on SF compared to blood neutrophils and macrophages. However, arginase-rich primary granules were not significantly exocytosed by SF neutrophils. Finally, we measured high levels of neutrophil chemoattractants (IL-8, G-CSF, and IL-1α) in SF compared to plasma. Conclusions Neutrophils are recruited to the joints of JIA patients and accumulate arginase on their surface, presumably released from tertiary, but not primary, granules. Arginase activity in the SF may lead to CD3 downregulation and T-cell inhibition, potentially in an effort to limit autoimmune complications.

Background Neutrophilic inflammation is a hallmark of cystic fibrosis (CF) lung disease. Prior studies from our group show that blood neutrophils undergo metabolic and functional adaptations upon entry into the CF lung lumen that causes them to become anabolic, while repressing bacterial killing function and enhancing degranulation. The recent advent of highly effective modulator therapy (HEMT) for the CFTR channel (mutated in CF) has improved patient outcomes, but it is unclear how HEMT impact CF lung neutrophilic inflammation. Method We used an organotypic model in which blood neutrophils are transmigrated through differentiated airway epithelial cells towards a chemoattractant control (leukotriene B4, LTB4), or airway supernatant from CF patients not on HEMT (CFASN) or on HEMT (CFMOD). We conducted phenotypic (FACS, CFU-assay) and metabolomic (metabolomics, 13C6-glucose tracing) analysis of the transmigrated neutrophils. Results CFMOD-recruited neutrophils contained ivacaftor (a HEMT drug), demonstrating exposure to the therapy. Compared to LTB4-recruited neutrophils, CFASN/CFMOD-recruited neutrophils showed increased AMP, GMP and intracellular and secreted metabolites related to citric acid cycle activity. 13C6-glucose flux indicated similar rates of extracellular glucose utilization in all transmigrated neutrophils, but higher total glycolysis to lactate in CFASN/CFMOD-transmigrated neutrophils. Conclusion Pathological metabolic adaptations of neutrophils following transmigration into CF airway fluid are not substantially altered by HEMT. Further studies are needed to assess the potential impact on functional adaptations by these cells. Supported by NIH (R56 HL150658), Cystic Fibrosis Foundation (CAMMAR21F0, TIROUV19G0), I3 Teams Research Award (Emory), and CF@LANTA, a component of Emory University and Children’s Healthcare of Atlanta.

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One Sentence Summary E3-ubiquitin ligase Hyd mediated ubiquitination of Akirin is required for its binding to NF-κBand the activation of a subset of target genes mediating the immune response against Gramnegative bacteria infection in Drosophila.. CC-BY-NC-ND 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/323170 doi: bioRxiv preprint first posted online May. 16, 2018; ABSTRACT Activation of inflammatory response is a tightly controlled process that is mediated by NF-κB pathways. Ubiquitinations are known to modulate the mammalian inflammatory response at all levels of the NF-κB pathways, but the mechanism involved in this ubiquitinmediated modulation of the inflammation is still not fully understood. Here, we used Drosophila genetics to identify E3-ubiquitin ligases involved in the innate immune response and explore their conservation in mammals. To this aim, we conducted an ex-vivo RNA interference screen of the E3 ubiquitin-ligases encoded by the fruit fly genome downstream of the Drosophila IMD pathway. This screen identified Hyperplastic Discs "Hyd" as acting genetically at the level of the NF-κB cofactor Akirin. Drosophila lacking Hyd failed to express the full set of anti-microbial peptides coding genes and succumbed to immune challenges. Remarkably, HyD-mediated K63-polyubiquitination of Akirin is required for the efficient binding of Akirin to the NF-κB transcription factor Relish. This Hyd-mediated interaction triggers the activation of a subset of Relish target genes, required in-vivo to survive immune challenges. We show further that Urb5, the mammalian homolog of Hyd, is also required in the NF-κB pathway. This study links the action of a E3-ubiquitin ligase to the activation of immune effector genes, deepening our understanding of the involvement of ubiquitination in inflammation and identifying a potential target for the control of inflammatory diseases.