IL-1R Signaling Research Articles

Single immunoglobulin and Toll‑interleukin‑1 receptor domain containing molecule protects against severe acute pancreatitis invitro by negatively regulating the Toll‑like receptor‑4 signaling pathway: A clinical and experimental study.

Single immunoglobulin and Toll-interleukin-1 receptor domain-containing molecule (SIGIRR) is a specific inhibitor of IL-1R and Toll-like receptor (TLR) signaling and considered a potential target for the treatment of inflammatory diseases. Pathogenic mechanisms associated with the TLR4 signaling pathway have a critical role in the development of severe acute pancreatitis (SAP). The aim of the present study was to determine the role of SIGIRR in the regulation of TLR4 signaling during the progression of SAP. Pancreatitis-associated ascitic fluid (PAAF) was collected from patients with SAP. Murine RAW264.7 macrophages were transfected with a SIGIRR overexpression plasmid and co-cultured with the PAAF from the donors in order to evaluate the effect of SIGIRR in vitro. The mRNA expression of TLR4, SIGIRR and other key downstream signaling molecules was quantified using semi-quantitative PCR with agarose gel electrophoresis. Furthermore, the levels of pro-inflammatory cytokines in the culture supernatant were detected using ELISA. In contrast to SIGIRR, the mRNA expression levels of TLR4, myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase-1 (IRAK-1) and TNF receptor-associated factor-6 (TRAF-6) were significantly increased in RAW264.7 cells following treatment with PAAF. Furthermore, TLR4, MyD88, IRAK-1 and TRAF-6 mRNA levels were significantly downregulated following SIGIRR overexpression and PAAF treatment in RAW264.7 cells. The levels of IL-2, IL-12, IL-17 and IFN-γ in the culture supernatant were also significantly decreased, while IL-10 levels were increased. Overall, SIGIRR negatively regulated the TLR4 signaling pathway to protect against the development of SAP in an in vitro model. Therefore, SIGIRR may represent a promising therapeutic target for SAP.

Loss of decay-accelerating factor triggers podocyte injury and glomerulosclerosis.

Kidney glomerulosclerosis commonly progresses to end-stage kidney failure, but pathogenic mechanisms are still poorly understood. Here, we show that podocyte expression of decay-accelerating factor (DAF/CD55), a complement C3 convertase regulator, crucially controls disease in murine models of adriamycin (ADR)-induced focal and segmental glomerulosclerosis (FSGS) and streptozotocin (STZ)-induced diabetic glomerulosclerosis. ADR induces enzymatic cleavage of DAF from podocyte surfaces, leading to complement activation. C3 deficiency or prevention of C3a receptor (C3aR) signaling abrogates disease despite DAF deficiency, confirming complement dependence. Mechanistic studies show that C3a/C3aR ligations on podocytes initiate an autocrine IL-1β/IL-1R1 signaling loop that reduces nephrin expression, causing actin cytoskeleton rearrangement. Uncoupling IL-1β/IL-1R1 signaling prevents disease, providing a causal link. Glomeruli of patients with FSGS lack DAF and stain positive for C3d, and urinary C3a positively correlates with the degree of proteinuria. Together, our data indicate that the development and progression of glomerulosclerosis involve loss of podocyte DAF, triggering local, complement-dependent, IL-1β-induced podocyte injury, potentially identifying new therapeutic targets.

Interleukin-1 promotes autoimmune neuroinflammation by suppressing endothelial heme oxygenase-1 at the blood\u2013brain barrier

The proinflammatory cytokine interleukin 1 (IL-1) is crucially involved in the pathogenesis of multiple sclerosis (MS) and its animal model experimental autoimmune encephalomyelitis (EAE). Herein, we studied the role of IL-1 signaling in blood–brain barrier (BBB) endothelial cells (ECs), astrocytes and microglia for EAE development, using mice with the conditional deletion of its signaling receptor IL-1R1. We found that IL-1 signaling in microglia and astrocytes is redundant for the development of EAE, whereas the IL-1R1 deletion in BBB-ECs markedly ameliorated disease severity. IL-1 signaling in BBB-ECs upregulated the expression of the adhesion molecules Vcam-1, Icam-1 and the chemokine receptor Darc, all of which have been previously shown to promote CNS-specific inflammation. In contrast, IL-1R1 signaling suppressed the expression of the stress-responsive heme catabolizing enzyme heme oxygenase-1 (HO-1) in BBB-ECs, promoting disease progression via a mechanism associated with deregulated expression of the IL-1-responsive genes Vcam1, Icam1 and Ackr1 (Darc). Mechanistically, our data emphasize a functional crosstalk of BBB-EC IL-1 signaling and HO-1, controlling the transcription of downstream proinflammatory genes promoting the pathogenesis of autoimmune neuroinflammation.

Glucose Induces IL-1α-Dependent Inflammation and Extracellular Matrix Proteins Expression and Deposition in Renal Tubular Epithelial Cells in Diabetic Kidney Disease.

Diabetes mellitus is linked with metabolic stress that induces cellular damage and can provoke renal inflammation and fibrotic responses that eventually lead to chronic kidney disease. Because the inflammasome, interleukin 1 (IL-1), IL-1α/IL-β, and IL-1R are central elements of kidney inflammation and pharmacological IL-1R antagonist (IL-1Ra) was shown to prevent or even reverse diabetic nephropathy (DN) in animal models, we explored the intrinsic expression of IL-1 molecules in kidney tissue of DN patients as regulators of renal inflammation. We used biopsies taken from DN patients and controls and show a high level of IL-1α expression in renal tubular epithelial cells, whereas both IL-1 agonistic molecules (i.e., IL-1α and IL-1β) were devoid of the glomeruli. Human proximal tubular kidney HK-2 cells exposed to high glucose (HG) gradually increase the expression of IL-1α but not IL-1β and induce the expression and deposition of extracellular matrix (ECM) proteins. We further demonstrate that in vitro ectopic addition of recombinant IL-1α in low glucose concentration leads to a similar effect as in HG, while supplementing excess amounts of IL-1Ra in HG significantly attenuates the ECM protein overexpression and deposition. Accordingly, inhibition of IL-1α cleaving protease calpain, but not caspapse-1, also strongly reduces ECM protein production by HK-2 cells. Collectively, we demonstrate that IL-1α and not IL-1β, released from renal tubular cells is the key inflammatory molecule responsible for the renal inflammation in DN. Our result suggests that the clinical use of IL-1Ra in DN should be promoted over the individual neutralization of IL-1α or IL-1β in order to achieve better blocking of IL-1R signaling.

Long Term Delta-9-tetrahydrocannabinol Administration Inhibits Proinflammatory Responses in Minor Salivary Glands of Chronically Simian Immunodeficieny Virus Infected Rhesus Macaques.

HIV/SIV-associated oral mucosal disease/dysfunction (HAOMD) (gingivitis/periodontitis/salivary adenitis) represents a major comorbidity affecting HIV patients on anti-retroviral therapy. Using a systems biology approach, we investigated molecular changes (mRNA/microRNA) underlying HAOMD and its modulation by phytocannabinoids (delta-9-tetrahydrocannabinol (∆9-THC)) in uninfected (n = 5) and SIV-infected rhesus macaques untreated (VEH-untreated/SIV; n = 7) or treated with vehicle (VEH/SIV; n = 3) or ∆9-THC (THC/SIV; n = 3). Relative to controls, fewer mRNAs were upregulated in THC/SIV compared to VEH-untreated/SIV macaques. Gene enrichment analysis showed differential enrichment of biological functions involved in anti-viral defense, Type-I interferon, Toll-like receptor, RIG-1 and IL1R signaling in VEH-untreated/SIV macaques. We focused on the anti-ER-stress anterior gradient-2 (AGR2), epithelial barrier protecting and anti-dysbiotic WAP Four-Disulfide Core Domain-2 (WFDC2) and glucocorticoid-induced anti-inflammatory TSC22D3 (TSC22-domain family member-3) that were significantly downregulated in oropharyngeal mucosa (OPM) of VEH-untreated/SIV macaques. All three proteins localized to minor salivary gland acini and secretory ducts and showed enhanced and reduced expression in OPM of THC/SIV and VEH/SIV macaques, respectively. Additionally, inflammation associated miR-21, miR-142-3p and miR-29b showed significantly higher expression in OPM of VEH-untreated/SIV macaques. TSC22D3 was validated as a target of miR-29b. These preliminary translational findings suggest that phytocannabinoids may safely and effectively reduce oral inflammatory responses in HIV/SIV and other (autoimmune) diseases.

Neuronal interleukin-1 receptors mediate pain in chronic inflammatory diseases.

Chronic pain is a major comorbidity of chronic inflammatory diseases. Here, we report that the cytokine IL-1β, which is abundantly produced during multiple sclerosis (MS), arthritis (RA), and osteoarthritis (OA) both in humans and in animal models, drives pain associated with these diseases. We found that the type 1 IL-1 receptor (IL-1R1) is highly expressed in the mouse and human by a subpopulation of TRPV1+ dorsal root ganglion neurons specialized in detecting painful stimuli, termed nociceptors. Strikingly, deletion of the Il1r1 gene specifically in TRPV1+ nociceptors prevented the development of mechanical allodynia without affecting clinical signs and disease progression in mice with experimental autoimmune encephalomyelitis and K/BxN serum transfer-induced RA. Conditional restoration of IL-1R1 expression in nociceptors of IL-1R1-knockout mice induced pain behavior but did not affect joint damage in monosodium iodoacetate-induced OA. Collectively, these data reveal that neuronal IL-1R1 signaling mediates pain, uncovering the potential benefit of anti-IL-1 therapies for pain management in patients with chronic inflammatory diseases.

Alterations in corneal biomechanics underlie early stages of autoimmune-mediated dry eye disease

Autoimmune-mediated dry eye disease is a pathological feature of multiple disorders including Sjögren's syndrome, lupus and rheumatoid arthritis that has a life-long, detrimental impact on vision and overall quality of life. Although late stage disease outcomes such as epithelial barrier dysfunction, reduced corneal innervation and chronic inflammation have been well characterized in both human patients and mouse models, there is little to no understanding of early pathological processes. Moreover, the mechanisms underlying the loss of cornea homeostasis and disease progression are unknown. Here, we utilize the autoimmune regulatory (Aire)-deficient mouse model of autoimmune-mediated dry eye disease in combination with genome wide transcriptomics, high-resolution imaging and atomic force microscopy to reveal a potential extracellular matrix (ECM)-biomechanical-based mechanism driving cellular and morphological changes at early disease onset. Early disease in the Aire-deficient mouse model is associated with a mild reduction in tear production and moderate immune cell infiltration, allowing for interrogation of cellular, molecular and biomechanical changes largely independent of chronic inflammation. Using these tools, we demonstrate for the first time that the emergence of autoimmune-mediated dry eye disease is associated with an alteration in the biomechanical properties of the cornea. We reveal a dramatic disruption of the synthesis and organization of the extracellular matrix as well as degradation of the epithelial basement membrane during early disease. Notably, we provide evidence that the nerve supply to the cornea is severely reduced at early disease stages and that this is independent of basement membrane destruction or significant immune cell infiltration. Furthermore, diseased corneas display spatial heterogeneity in mechanical, structural and compositional changes, with the limbal compartment often exhibiting the opposite response compared to the central cornea. Despite these differences, however, epithelial hyperplasia is apparent in both compartments, possibly driven by increased activation of IL-1R1 and YAP signaling pathways. Thus, we reveal novel perturbations in corneal biomechanics, matrix organization and cell behavior during the early phase of dry eye that may underlie disease development and progression, presenting new potential targets for therapeutic intervention.

591-P: Macrophage IL-1beta–Dependent VEGF-A Expression Is Required for Inflammatory Arteriogenesis and Wound Healing in Type 2 Diabetes

Impaired wound healing in patients with diabetes mellitus (DM) is a major source of morbidity and mortality. Current treatments for diabetic wounds are modestly effective at promoting healing. Recently, we identified an important role for inflammatory macrophage VEGF-A production in consequent angiogenesis/arteriogenesis required for adequate wound healing. We sought to understand the impact of diabetes on inflammatory angiogenesis and consequent healing. We hypothesized that diabetes mellitus (DM) results in reduced inflammatory macrophage VEGF-A expression with consequent impairments in angiogenesis-dependent wound healing. Mice with experimental diabetes demonstrated profound delays both in wound healing using a dermal punch biopsy model and in perfusion recovery using the femoral artery ligation model of hind limb ischemia. VEGF-A expression is highly upregulated in “classic inflammatory” macrophages relative to “alternatively activated” or wound healing macrophages. This is supported by a strong association between IL-1β and VEGF-A expression. In order to understand whether this relationship between IL-1β and VEGF-A was causal, we generated a novel mouse with a LoxP flanked IL-1β allele to create an inducible macrophage deletion of IL-1β. Myeloid IL-β deletion resulted in severely impaired macrophage VEGF-A expression and consequent decreases in angiogenesis and wound healing, consistent with the experimental model of DM. Moreover, macrophages from diabetic mice demonstrated reduced expression of IL-1R signaling complex components along with consequent reductions in VEGF-A expression. Defining inflammatory macrophages as key, early drivers of angiogenesis supports a paradigm shift away from inflammation-suppression for adequate wound healing, allowing for macrophage reprograming strategies that promote appropriate inflammation-dependent healing responses. Disclosure C.S. Mantsounga: None. Funding National Institutes Health (1R01HL139795)

Inflammation impairs IL-2R signaling in regulatory T cells

Abstract Low-dose IL-2 is a promising therapy to boost Tregs in autoimmune diseases. Inflammation accompanying autoimmunity destabilizes Tregs to diminish their function. Here we explored how the extent of acute inflammation induced by LPS and poly(I:C) and of chronic inflammation induced by immunization with MOG and pertussis toxin (MOG/PT) affects IL-2R signaling in Tregs. IL-2-dependent pSTAT5 was significantly, but transiently, reduced in Tregs ex vivo from C57BL/6 mice exposed to LPS and MOG/PT, but not poly(I:C). LPS and PT, but not poly(I:C), induced inflammation through TLR4. IL-2-dependent pSTAT5 was also decreased in WT mice treated with only PT or in TLR4 Treg-conditional knockout mice treated with LPS. Thus, impaired IL-2R signaling is likely dependent on monocyte-derived inflammatory mediators through TLR4 signaling. LPS also decreased Treg proliferation as measured by Ki67 and increased CD69 expression. In contrast, these changes were not seen after treatment with poly(I:C) and MOG/PT, consistent with inflammation affecting Tregs independent of lower IL-2R signaling. When Tregs from LPS-treated mice were stimulated with IL-2 in vitro, pSTAT5 was induced, indicating that Tregs are not intrinsically unable to respond to IL-2. RNAseq of Tregs from LPS-treated mice revealed substantial changes in their transcriptome, suggesting a potential complex mechanism that impairs IL-2R signaling and other activities in Tregs in vivo. Our data support a model where inflammation lowers IL-2R signaling in Tregs by decreased IL-2 in the Treg microenvironment and causes other induced changes in Tregs to lower their function. Thus, a beneficial feature of low-dose IL-2 therapy may be to counteract negative effects of inflammation on Tregs.

Oral epithelial IL-22/STAT3 signaling licenses IL-17-mediated immunity to oral mucosal candidiasis

Abstract Oropharyngeal candidiasis (OPC) is an opportunistic infection of the oral mucosa caused by the commensal fungus Candida albicans. IL-17 and IL-22 both mediate antifungal immunity yet activate distinct downstream signaling pathways. While much is known about IL-17-dependent immunity in OPC, the activities of IL-22 are less well delineated. We show that induction of Il22 is independent of Dectin-1, CARD9 and aryl hydrocarbon receptor (AhR) and is driven by IL-23 and the C. albicans pore forming peptide candidalysin. Despite similar induction requirements and cellular sources, IL-22 and IL-17 function non-redundantly during OPC and exert opposing roles in neutrophil recruitment. The IL-22 and IL-17 receptors are required in anatomically distinct locations; loss of IL-22RA1 in the oral basal epithelial layer (BEL) but not the suprabasal epithelial layer (SEL) causes susceptibility to OPC, whereas IL-17RA is needed in the SEL. Our data reveal that IL-22 is a major activator of STAT3 in the BEL during OPC. Moreover, loss of STAT3 in the BEL but not the SEL renders mice susceptible to OPC. Transcriptional profiling of RNASeq data linked IL-22/STAT3 to oral epithelial cell proliferation and survival, but also, unexpectedly, to driving an IL-17 gene signature. We show that IL-22 acts on the BEL to replenish the IL-17RA-expressing SEL, thereby restoring the ability of the oral epithelium to respond to IL-17. Consequently, IL-22 signaling in BEL ‘licenses’ IL-17R signaling in the oral epithelium, revealing spatially distinct yet cooperative activities of IL-22 and IL-17 in oral candidiasis. This work also suggests that oral thrush in Jobs’ syndrome patients may be caused by STAT3 impairments in the oral epithelium, not just Th17 cells.

MicroRNA-146a limits tumorigenic IL-17-mediated inflammation in colorectal cancer

Abstract Interleukin-17 (IL-17) is a major inflammatory cytokine implicated in colorectal cancer (CRC) development. However, the mechanisms that control tumorigenic IL-17 signaling remain unclear. Recently, expression changes and polymorphisms in the small non-coding RNA, microRNA-146a (miR-146a), have been associated with clinical outcomes in inflammatory bowel disease and CRC patients. Here, we identified a novel role for miR-146a as a major negative regulator of colonic inflammation and tumorigenesis via modulation of IL-17 responses. MiR-146a-deficient mice are highly susceptible to both colitis-associated and sporadic CRC, and present with enhanced tumorigenic IL-17 signaling. Within myeloid cells, miR-146a targets RIPK2, an intermediate in NOD2 signaling, to limit myeloid cell-derived IL-17-inducing cytokines and restrict colonic IL-17 levels. Accordingly, myeloid cell-specific deletion of miR-146a leads to CRC susceptibility. Moreover, within intestinal epithelial cells (IECs), miR-146a targets TRAF6, an intermediate in IL-17R signaling, to restrict IEC responsiveness to IL-17. MiR-146a within IECs further suppresses CRC by targeting PTGES2, an enzyme for PGE2 synthesis. IEC-specific deletion of miR-146a therefore confers marked CRC susceptibility. Importantly, preclinical administration of miR-146a mimic can ameliorate colonic inflammation and CRC. In conclusion, miR-146a prevents CRC by two interlinked mechanisms: 1) by limiting myeloid cell-mediated inflammatory IL-17 production; and 2) by inhibiting tumorigenic IL-17R signaling in IECs. Overexpression of miR-146a may be a promising therapeutic approach for CRC to limit multiple pathways converging on tumorigenic IL-17 signaling.

IL-4R signaling mediated polarization of neutrophils promoted monocytes derived alveolar macrophages function

Abstract We have reported that alternatively activated neutrophils (N2) enhance lung anti-helminth trained macrophage effector function in response to infection with N. brasiliensis(Fei Chen, et al., NI, 2014). However, it is not clear how these neutrophils develop and what lung macrophage subsets actually mediate resistance. We report here that IL-4R signaling is required for the polarization of neutrophils within 2 days after N. brasiliensisinfection and that this characteristic N2 phenotype can develop in Rag−/− but not Rag2cg−/− mice, suggesting a role for innate lymphoid cells in initiating N2 cell differentiation. N2 neutrophils further showed significant proliferative capacity and oxidative phosphorylation at day 2 after N. brasiliensisinfection. Using CD45 congenic transfers and fate mapping Cx3cr1creER-YFP/+R26tdTomato/+ mice, we further show that monocyte-derived alveolar macrophages are the major effector macrophage subset mediating parasite killing through Arginase 1 dependent mechanisms. In vivo depletion of neutrophils with anti-Ly6G Ab blocked the development of this anti-helminth pulmonary macrophage subset and RNAseq analyses show marked differences from tissue resident alveolar macrophages. These studies demonstrate heterogeneity in lung macrophage and neutrophil subsets during helminth infection resulting in distinct activation pathways required for helminth resistance.

Blimp1 suppresses proliferation in human regulatory T cells as a potential homeostasis regulator

Abstract B lymphocyte-induced maturation protein 1 (Blimp1), encoded by the PRDM1 gene, is a multifunctional transcriptional regulator that has established roles in mouse T cells. However, the mechanisms underlying its function in the proliferation, suppressive function, and homeostasis of human regulatory T cells (Tregs) are not known. To directly evaluate the role of Blimp1 in human Tregs, Blimp1α, the active isoform of Blimp1 that has dominant expression, was knocked out by Crispr-Cas9 using dual-targeting sgRNAs. High knockout efficiency was verified at DNA, RNA, and protein level by T7E1, RT-PCR, and Western blot analyses, respectively. Knockout of Blimp1α in cultured human Tregs resulted in increased cell number, upregulated Ki67 expression, and enhanced [3H]thymidine incorporation, consistent with a normal inhibitory role of Blimp1α in the proliferation of human Tregs. Compared with control cells, Blimp1α-knockout Tregs showed high CD25 expression and enhanced tyrosine phosphorylation of STAT5 (pSTAT5), suggesting a potential role of Blimp1 in regulation of IL-2R signaling. In vitro suppression assay showed that knockout of Blimp1α slightly decreased the suppressive function of Tregs. Knockout of Blimp1α also increased the expressions of Tigit and CD62L, but decreased the expressions of Foxp3, CD73, and Bcl-2, suggesting that Blimp1α may contribute to maintaining Treg identity. Collectively, our results are consistent with a model whereby Blimp1α regulates homeostasis of human Tregs by constraining proliferation in part by limiting IL-2R signaling while promoting Treg function and identity.

Characterization of IL7R α in Xenopus tropicalis: implications for the evolution of cytokine signaling pathways

Abstract Signaling through the IL7 receptor is essential for development of B and T cells in humans and mice, as evidenced by the SCID-like phenotype seen in the absence of IL7/IL7 receptor signaling. The IL7R signaling system is active in lower vertebrates but has not previously been examined in Xenopus frogs. The IL7R is a heterodimer comprised of the IL7R α chain and the “common” γ chain that is shared by other IL2 family cytokines that utilize the JAK-STAT pathway. We have recently cloned the IL7R α cDNA from Xenopus tropicalis. Comparison of the predicted amino acid sequence of the Xenopus IL7R α indicates closest homology with the zebrafish IL7R α, and significant homology with other characterized IL7R α including those from humans, mice, chickens, alligators, primitive caecilians, green sea turtles, and anole lizards. Analysis of the predicted amino acid sequence indicates an intact WSXWS motif (common to all IL7R family members). The Box 1 motif, normally required for JAK interaction, has an asparagine replacing aspartic acid (similar variations are found in the zebrafish and anole lizard IL7R α sequences). There are at least five potential N glycosylation sites. There is a 16 amino acid insert unique to the Xenopus sequence that is not found in the reported IL7R α sequence of any other animal that we have examined. Our attempts to identify an IL-7 gene in the Xenopus genome have been unfruitful. Synteny analysis indicate there is no gene with homology to IL-7 at the expected position in the Xenopus genome. Interestingly, we have identified a transposon in place of the IL-7 gene at the expected position. The distribution of IL7R α in various organs of X. tropicalis is now being examined, and as well predictions of the 3D structure of the Xenopus IL7R α protein.

Therapeutic hypothermia rescues severe acute lung injury (ALI) by IL-1β-induced neutrophil extracellular traps (NETs) formation through down-regulation of Gasdermin D

Abstract ARDS is a serious ALI defined by the acute onset of non-cardiogenic pulmonary edema and hypoxemia. We have previously published that NLRP3 inflammasome is required to develop hypoxemia in LPS plus mechanical ventilator (MV) two-hit-induced ALI. We initially observed that, despite equal neutrophil infiltration, severe alveolar leakage and hypoxemia were not observed in Il1r1KO mice, confirming that NLRP3 inflammasome is critical in this two-hit induced hypoxemia. However, this hypoxemia was significantly attenuated in neutrophil-depleted mice. Furthermore, we found that NETs formations were significantly attenuated in Il1r1KO mice. These data demonstrated that IL-1R signaling plays a regulating role in neutrophil functionality in the alveoli, and not their infiltration. We next examined whether NETs formation contributed to develop hypoxemia in two-hit ALI. by Pad4 inhibitor and DNase I. Both treatments improved the development of hypoxemia. In the course of experiments, we serendipitously found that body temperature is a critical factor regulating severe hypoxemia. Therapeutic hypothermia at 32°C during MV rescued development of hypoxemia with significantly attenuated alveolar leakage, reduced IL-1β secretion, and less NETs formation. 32°C condition inhibited IL-1β secretion by Mϕ as well as NETs induction. Unexpectedly, we observed similar Caspase-1 activities in LPS-primed Mϕ stimulated with ATP or nigericin at 32°C while IL-1βsecretion was significantly reduced. We found decreased GSDMD expression and unsecreted mature IL-1β at 32 °C treatment. Overall our data suggests that hypothermia suppresses ALI development by inhibiting IL-1β secretion and NETs formation through down-regulation of GSDMD.

B Cell derived IL-4 promotes IL-10 secreting TIM-1+ regulatory B cell expansion and regulates Th2 T cell responses

Abstract TIM-1+ B cells are enriched for IL-10+ B regulatory cells (Bregs). However, TIM-1+ B cells are also enriched for IL-4+ B effector 2 cells (Be2). Tolerogenic α-TIM-1 (RMT1-10) prolongs islet allograft survival (GS) by expanding (IL-10+) Bregs. Interestingly, IL-4KO and IL-4Rα-KO mice are deficient in Bregs and are not responsive to αTIM-1. To establish the importance of IL-4R signaling, and the source of IL-4 for Breg induction, we made tamoxifen (TAM) inducible BALB/c hCD20Cre.ERT2+/− X IL-4Rαfl/fl (B-IL-4RαKO) and hCD20Cre.ERT2+/− X IL-4/13fl/fl mice (B-IL-4KO) to respectively delete IL-4Rα or IL-4/IL-13 in B cells. After TAM, αTIM-1 treatment of alloimmunized B-IL-4RαKO mice had ↓ TIM-1+ B cells (50%), Be2 (50%) and Bregs (40%) vs. IL-4Rαfl/fl littermate controls and a concomittant 33%↑ in IFNγ+ and 33% ↓ in IL-10+ CD4 T cells, and a 20%↑ in IFNγ+ CD8 T cells vs. controls. Finally, α–TIM-1 led to >100d GS in 75% of control mice, but failed to improve GS in B-IL-4RαKO mice (MST 20d, p=0.03). Similarly, TAM treated B-IL-4KO mice had a 3x ↓ in Be2 and Bregs vs. IL-4fl/fl controls, and a 6x drop in IL-4+ CD4 T cells. Importantly, B-IL-4KO mice rejected islets more rapidly (MST 20d) than controls (MST 31d, p=0.04), and α–TIM-1 failed to improve B-IL-4KO GS (MST 17d) vs. controls (MST >100d). In contrast, in Th2 dominant OVA-induced allergic airway disease (AAD), B-IL-4KO mice had 3x decrease in total lymphocyte infiltrate, 5x decrease CD4 T cells and 8x decrease eosinophils in BAL fluid vs. controls. CD4+ T cells from whole lung tissue produced 2x less IL-4 and 3x less IL-5 vs. controls. Hence, IL-4 production by B cells is important for both Breg induction and Th2 polarization and absence of B cell IL-4/IL-13 promotes allograft rejection yet reduces AAD.

Dysregulation of type I IFN and IL-4R signaling regulates the development of BCR VH and VL repertoire encoding anti-Sm autoantibodies in SLE

Abstract The most specific predictor for systemic lupus erythematosus (SLE) and lupus nephritis (LN) is the presence of anti-Smith (Sm) autoAb. Mechanisms leading to selection bias for anti-Sm development is unclear. We found that anti-Sm levels in SLE correlated with increased endogenous IFN-β and interferon-stimulated genes (ISGs) in combination with low (IL-4R) network genes. Using flow cytometry analysis of 47 SLE patients, we found that anti-Sm+ patients exhibited an increased ratio of IFN-β+ activated naive (aNAV) B cells and a decrease in IL-4R+ resting naive (rNAV) B cells. Using a combined 10X single-cell transcriptomics and BCR usage analysis, anti-Sm+LN+ISGhiIL4Rlopatients (Sm+) were compared with anti-Sm−LN− ISGloIL4Rhi patients (Sm−). BCR heavy chain analysis showed that while B cells from Sm+ patients exhibit greater usage of IGHV1-2, IGHV4-34, and IGHV4-39 genes, Sm− SLE patients exhibit greater usage of IGHV3-30, IGHV3-33, and IGHV4-31 genes. Preferential Ig light chain usage for ISGhi B cells was IGLV1-51, IGLV3-21, IGLV6-57, IGKV1-5, IGKV3-15 and IGKV 6-21. Dominant heavy-light chain pair usages for the Sm+ patients were IGHV4/IGKV1 and IGHV4/IGKV3. IGHV4-34, which is intrinsically autoreactive and the target of the 9G4 anti-idiotype Ab, was found to be expressed by the ISGhiIL4Rlo B cells derived from the same Sm+ patients. The results indicated that increased levels of ISGs and decreased levels of IL-4R pathway genes are the two dominant signatures and the underlying mechanisms leading to selection and activation of anti-Sm+ B cells. Verification that the B-cell IFN-β and IL-4R phenotypes are associated with race (AA vs. EA), repertoire (anti-Sm+) and disease (LN+) would lay the basis for targeted rational therapies.

Cul5 E3 ubiquitin ligase limits Th2 and Th9 differentiation by regulating IL4 receptor level in CD4 T cells

Abstract Allergic asthma is a chronic airway disease characterized by increased airway resistance and airway remodeling. It is well established that increased Th2 and Th9 cells is responsible for this phenotype, the mechanisms restricting these cell numbers are poorly understood. Here we show that the E3 ubiquitin ligase, Cul5, restricts Th2 and Th9 differentiation and prevents allergic asthma. We found that Cul5 activity measured by neddylation is increased following CD4 T cell stimulation. We generated mice lacking Cul5 in T cells that showed mild Th2 inflammation in lung specifically. Upon house dust mite exposure these mice exhibited increased eosinophilia, goblet cell hyperplasia, and airways remodeling. Mechanistically, we show that in activated T cells, Cul5 associated with cytokine-induced SH2 protein (CIS), a SOCS-box protein. CIS act as a substrate receptor for Cul5 E3 ubiquitin complex and limits IL-4 receptor level on the surface, that correlated with increased Th2 and Th9 differentiation and/or persistence in vivo. These data support that Cul5 works with CIS to attenuate IL-4R signaling and reduce susceptibility to allergic asthma.

Endogenous IL-33 contributes to Treg expansion induced by IL-2/CD25, a novel IL-2 biologic developed for low-dose IL-2 therapy

Abstract Low-dose IL-2 therapy is being advanced in patients with autoimmunity by its ability to increase Tregs. A drawback of IL-2, however, is its short-half life and potential off-target immuno-stimulatory effects. To circumvent these limitations, we developed a longer-lasting mouse IL-2/CD25 fusion protein with greater selectivity toward Tregs. IL-2/CD25 is more effective than an equivalent amount of IL-2 in controlling diabetes of NOD mice. Furthermore, compared to other tissues, IL-2/CD25 more readily expanded pancreatic Tregs, suggesting a co-factor that acts with IL-2/CD25. We considered IL-33 as a possible candidate as it also promotes Treg expansion. In this regard, low-dose IL-2/CD25 selectively induced the IL-33R (ST2) on NOD pancreatic Tregs. Correspondingly, ST2 was not substantially induced in Tregs with impaired IL-2R signaling. More importantly, IL-33 supported the expansion of wild-type Tregs, but not Tregs with impaired IL-2R signaling. Thus, Treg expansion by IL-33 depends on IL-2. Reciprocally, Treg expansion in the pancreas by IL-2/CD25 required IL-33 because this expansion was suboptimal in IL-33-deficient B6 mice but was maximal when IL-33 was sequentially administered with IL-2/CD25. However, IL-2/CD25 and IL-33 did not act synergistically for Treg expansion in the pancreas of NOD mice, as IL-33 or IL-2/CD25 were similarly effective individually. This result may reflect that other inflammatory mediators or co-factors associated with the NOD pancreas also influence the Treg response to IL-2/CD25 and IL-33. Overall, these data indicate that tissue-derived IL-33 may contribute to the overall outcome of low-dose IL-2 therapy by supporting Treg expansion during therapy of autoimmune diseases.

Limited application of IL-2/CD25 leads to persistent IL-2R signaling that promotes gene expression for Treg expansion

Abstract Low-dose IL-2 expands Tregs to suppress autoimmunity. However, IL-2 has a short half-life that necessitates frequent administration to maintain increased Treg levels. To improve low-dose IL-2 therapy, we developed an IL-2-based fusion protein, mouse IL-2/CD25, that substantially increases IL-2 half-life to more effectively boost Tregs that controls diabetes in NOD mice. To provide a molecular underpinning for the Treg response to IL-2/CD25, transcriptional profiling was performed by RNAseq for purified Tregs from C57BL/6 mice that received an equivalent amount of IL-2 or IL-2/CD25. Initially, at 4 hr post-injection, differential gene expression analysis revealed a large overlap in significant genes modulated by IL-2 or IL-2/CD25. In striking contrast, at 16 and 72 hr post-injection, only IL-2/CD25 persistently modulated gene expression, with the largest number of IL-2-dependent genes detected at 72 hrs. Canonical pathway analysis revealed many interrelated pathways that control cell growth and signaling. This gene expression pattern by IL-2/CD25, but not IL-2, was also associated with extended STAT5 activation and expansion of Tregs with molecular and phenotypic changes corresponding to activated Tregs. IL-2/CD25 also enhanced gene expression associated with glycolysis and the mitochondrial electron transport chain. Seahorse analysis confirmed that IL-2/CD25 shifted Tregs to a more energetic metabolic phenotype due to an increase in mitocondrial ATP. Collectively, these findings demonstrate that a single injection of IL-2/CD25 persistently stimulates Tregs to activate gene expression to enhance Treg metabolism to fulfill the bio-energetic and biosynthetic demands of activation and proliferation.