Interferon-λ enhances adaptive mucosal immunity by boosting release of thymic stromal lymphopoietin.

Interferon-λ (IFN-λ) acts on mucosal epithelial cells and thereby confers direct antiviral protection. In contrast, the role of IFN-λ in adaptive immunity is far less clear. Here, we report that mice deficient in IFN-λ signaling exhibited impaired CD8+ T cell and antibody responses after infection with a live-attenuated influenza virus. Virus-induced release of IFN-λ triggered the synthesis of thymic stromal lymphopoietin (TSLP) by M cells in the upper airways that, in turn, stimulated migratory dendritic cells and boosted antigen-dependent germinal center reactions in draining lymph nodes. The IFN-λ-TSLP axis also boosted production of the immunoglobulins IgG1 and IgA after intranasal immunization with influenza virus subunit vaccines and improved survival of mice after challenge with virulent influenza viruses. IFN-λ did not influence the efficacy of vaccines applied by subcutaneous or intraperitoneal routes, indicating that IFN-λ plays a vital role in potentiating adaptive immune responses that initiate at mucosal surfaces.

Lipid-Droplet Formation Drives Pathogenic Group 2 Innate Lymphoid Cells in Airway Inflammation.

Innate lymphoid cells (ILCs) play an important role in the control and maintenance of barrier immunity. However, chronic activation of ILCs results in immune-mediated pathology. Here, we show that tissue-resident type 2 ILCs (ILC2s) display a distinct metabolic signature upon chronic activation. In the context of allergen-driven airway inflammation, ILC2s increase their uptake of both external lipids and glucose. Externally acquired fatty acids are transiently stored in lipid droplets and converted into phospholipids to promote the proliferation of ILC2s. This metabolic program is imprinted by interleukin-33 (IL-33) and regulated by the genes Pparg and Dgat1, which are both controlled by glucose availability and mTOR signaling. Restricting dietary glucose by feeding mice a ketogenic diet largely ablated ILC2-mediated airway inflammation by impairing fatty acid metabolism and the formation of lipid droplets. Together, these results reveal that pathogenic ILC2 responses require lipid metabolism and identify ketogenic diet as a potent intervention strategy to treat airway inflammation.

A mouse-adapted model of SARS-CoV-2 to test COVID-19 countermeasures.

Coronaviruses are prone to transmission to new host species, as recently demonstrated by the spread to humans of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic1. Small animal models that recapitulate SARS-CoV-2 disease are needed urgently for rapid evaluation of medical countermeasures2,3. SARS-CoV-2 cannot infect wild-type laboratory mice owing to inefficient interactions between the viral spike protein and the mouse orthologue of the human receptor, angiotensin-converting enzyme 2 (ACE2)4. Here we used reverse genetics5 to remodel the interaction between SARS-CoV-2 spike protein and mouse ACE2 and designed mouse-adapted SARS-CoV-2 (SARS-CoV-2 MA), a recombinant virus that can use mouse ACE2 for entry into cells. SARS-CoV-2 MA was able to replicate in the upper and lower airways of both young adult and aged BALB/c mice. SARS-CoV-2 MA caused more severe disease in aged mice, and exhibited more clinically relevant phenotypes than those seen in Hfh4-ACE2 transgenic mice, which express human ACE2 under the control of the Hfh4 (also known as Foxj1) promoter. We demonstrate the utility of this model using vaccine-challenge studies in immune-competent mice with native expression of mouse ACE2. Finally, we show that the clinical candidate interferon-λ1a (IFN-λ1a) potently inhibits SARS-CoV-2 replication in primary human airway epithelial cells in vitro-both prophylactic and therapeutic administration of IFN-λ1a diminished SARS-CoV-2 replication in mice. In summary, the mouse-adapted SARS-CoV-2 MA model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN-λ1a as a treatment for human COVID-196.

Interleukin-21-Producing CD4(+) T Cells Promote Type 2 Immunity to House Dust Mites.

Asthma is a T helper 2 (Th2)-cell-mediated disease; however, recent findings implicate Th17 and innate lymphoid cells also in regulating airway inflammation. Herein, we have demonstrated profound interleukin-21 (IL-21) production after house dust mite (HDM)-driven asthma by using T cell receptor (TCR) transgenic mice reactive to Dermatophagoides pteronyssinus 1 and an IL-21GFP reporter mouse. IL-21-producing cells in the mediastinal lymph node (mLN) bore characteristics of T follicular helper (Tfh) cells, whereas IL-21(+) cells in the lung did not express CXCR5 (a chemokine receptor expressed by Tfh cells) and were distinct from effector Th2 or Th17 cells. Il21r(-/-) mice developed reduced type 2 responses and the IL-21 receptor (IL-21R) enhanced Th2 cell function in a cell-intrinsic manner. Finally, administration of recombinant IL-21 and IL-25 synergistically promoted airway eosinophilia primarily via effects on CD4(+) lymphocytes. This highlights an important Th2-cell-amplifying function of IL-21-producing CD4(+) T cells in allergic airway inflammation.

Characterisation of the pruritus responses and pruritic behaviours in an interleukin 31-induced canine model of pruritus.

BACKGROUND: Intravenous administration of interleukin (IL)-31 in healthy dogs has been used as a model to assess antipruritic drugs. However, there is no known in-depth characterisation of pruritic behaviours, and the repeatability of the IL-31-induced pruritus in the individual dogs is currently unknown. OBJECTIVES: To evaluate the immediate/delayed pruritus responses and the pruritic behaviours observed in the IL-31-induced pruritic model in healthy dogs after repeated IL-31 injections. ANIMALS: Fifteen healthy laboratory beagles. METHODS: All dogs were video-recorded for 270 min after two intravenous recombinant IL-31 injections (1.75 µg/kg) and vehicle (phosphate-buffered saline, control) injections, respectively; interventions were randomised and performed with a 2 week wash-out period. Two blinded investigators reviewed the pruritic behaviours of all video recordings. RESULTS: Both canine IL-31 (IL-31_01, IL-31_02) injections significantly increased pruritic seconds and categorical minutes ('YES'/'NO' behaviour per discrete 1 min interval) in healthy dogs compared with both vehicle groups (Vehicle_01, Vehicle_02). The second intravenous canine IL-31 (IL-31_02) administered 14 days after the first IL-31 injection induced a significant increase in pruritic seconds (p = 0.021) and not pruritic categorical minutes (p = 0.231). An increase in pruritic seconds was observed in both IL-31 groups in the first 30 min post-administration, while there was no significant difference between IL-31 and vehicle groups. CONCLUSIONS AND CLINICAL RELEVANCE: In conclusion, intravenous IL-31 reproducibly induces itch responses in dogs. Future evaluations of the canine IL-31 pruritic model should assess total pruritic behaviours in seconds rather than using a biased 'YES/NO' behaviour per 1 min scoring system.

Induction of innate lymphoid cell-derived interleukin-22 by the transcription factor STAT3 mediates protection against intestinal infection.

Inhibitors of the transcription factor STAT3 target STAT3-dependent tumorigenesis but patients often develop diarrhea from unknown mechanisms. Here we showed that STAT3 deficiency increased morbidity and mortality after Citrobacter rodentium infection with decreased secretion of cytokines including IL-17 and IL-22 associated with the transcription factor RORγt. Administration of the cytokine IL-22 was sufficient to rescue STAT3-deficient mice from lethal infection. Although STAT3 was required for IL-22 production in both innate and adaptive arms, by using conditional gene-deficient mice, we observed that STAT3 expression in RORγt(+) innate lymphoid cells (ILC3s), but not T cells, was essential for the protection. However, STAT3 was required for RORγt expression in T helper cells, but not in ILC3s. Activated STAT3 could directly bind to the Il22 locus. Thus, cancer therapies that utilize STAT3 inhibitors increase the risk for pathogen-mediated diarrhea through direct suppression of IL-22 from gut ILCs.

Therapeutic effects of vitamin D and IL-22 on methotrexate-induced mucositis in mice.

Mucositis is a common side effect of cancer therapies and transplant conditioning regimens. Management of mucositis involves multiple approaches from oral hygiene, anti-inflammatory, anti-apoptotic, cytoprotective, and antioxidant agents, to cryo-therapy, physical therapy, and growth factors. There is room for novel, affordable treatment options, or improvement of currently available therapies. Vitamin D has been shown to regulate mucosa-resident cell populations such as Th17 or innate lymphoid cells and critical mucosal cytokine IL-22; however, their therapeutic potential has not been put to test in preclinical mouse models. In this study, we aimed to test the therapeutic potential of vitamin D injections and IL-22 overexpression in a murine model of chemotherapy-induced mucositis. Balb/c mice were given daily intraperitoneal injections of vitamin D. Mucositis was induced by methotrexate. Another group received IL-22 plasmid via hydrodynamic gene delivery. Weight loss and intestinal histopathology, intestinal levels of cytokines IL-22, IL-17A, GM-CSF, IL-23, IFN-γ, TNF-α, and IL-10, and number of intestinal lamina propria B cell, neutrophil, and total innate lymphoid cells were quantified. Daily vitamin D injections ameliorated intestinal inflammation and elevated intestinal IL-22 levels compared with control groups. Temporal overexpression of IL-22 by hydrodynamic gene delivery slightly increased intestinal IL-22 but failed to confer significant protection from mucositis. To our knowledge, this is the first experimental demonstration in an animal model of mucositis of therapeutic use of vitamin D and IL-22 supplementation and our results with vitamin D suggest it may have merit in further trials in human mucositis patients.

Interleukin-35 inhibits lipopolysaccharide-induced endothelial cell activation by downregulating inflammation and apoptosis.

Inflammation is an essential factor contributing to sepsis-induced endothelial cell (EC) activation. Interleukin-35 (IL-35) is an anti-inflammatory/immunosuppressive cytokine that exerts protective effects on many inflammatory diseases. In this study, we investigated the effects of IL-35 on lipopolysaccharide (LPS)-induced EC activation and the potential underlying mechanism. Human umbilical vein endothelial cells (HUVECs) were incubated with LPS (1 µg/ml) for 24 h and then cocultured with different concentrations (0, 1, 10, or 100 ng/ml) of recombinant human IL-35 (rhIL-35) for 12 h. Flow cytometry analysis revealed that IL-35 inhibited LPS-induced HUVEC apoptosis in a dose-dependent manner. RT-qPCR and Western blot analyses showed significantly higher mRNA and protein levels of the adhesion molecules intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and the inflammatory factors IL-6 and IL-8 in the LPS group than in the control group. These changes were alleviated by IL-35 treatment, suggesting that IL-35 protects ECs by downregulating inflammation. Furthermore, IL-35 induced signal transducer and activator of transcription 1 (STAT1) and STAT4 activation and promoted their interaction. Blocking STAT1 or STAT4 expression by fludarabine (STAT1 inhibitor) treatment or siRNA-STAT4-interfering fragment transfection inhibited the protective effect of IL-35 on ECs. Moreover, we observed a similar protective effect of IL-35 treatment on ECs in a mouse sepsis model induced by intraperitoneal LPS injection. This study indicated that IL-35 exerts anti-inflammatory and antiapoptotic effects on LPS-induced EC activation by activating the STAT1 and STAT4 signaling pathways.

Multiparametric magnetic resonance imaging/magnetic resonance elastography assesses progression and regression of steatosis, inflammation, and fibrosis in alcohol-associated liver disease.

BACKGROUND: Magnetic resonance imaging (MRI) and MRI-based elastography (MRE) are the most promising noninvasive techniques in assessing liver diseases. The purpose of this study was to evaluate an advanced multiparametric imaging method for staging disease and assessing treatment response in realistic preclinical alcohol-associated liver disease (ALD). METHODS: We utilized four different preclinical mouse models in our study: Model 1-mice were fed a fast-food diet and fructose water for 48 weeks to induce nonalcoholic fatty liver disease; Model 2-mice were fed chronic-binge ethanol (EtOH) for 10 days or 8 weeks to induce liver steatosis/inflammation. Two groups of mice were treated with interleukin-22 at different time points to induce disease regression; Model 3-mice were administered CCl4 for 2 to 4 weeks to establish liver fibrosis followed by 2 or 4 weeks of recovery; and Model 4-mice were administered EtOH plus CCl4 for 12 weeks. Mouse liver imaging biomarkers including proton density fat fraction (PDFF), liver stiffness (LS), loss modulus (LM), and damping ratio (DR) were assessed. Liver and serum samples were obtained for histologic and biochemical analyses. Ordinal logistic regression and generalized linear regression analyses were used to model the severity of steatosis, inflammation, and fibrosis, and to assess the regression of these conditions. RESULTS: Multiparametric models with combinations of biomarkers (LS, LM, DR, and PDFF) used noninvasively to predict the histologic severity and regression of steatosis, inflammation, and fibrosis were highly accurate (area under the curve > 0.84 for all). A three-parameter model that incorporates LS, DR, and ALT predicted histologic fibrosis progression (r = 0.84, p < 0.0001) and regression (r = 0.79, p < 0.0001) as measured by collagen content in livers. CONCLUSION: This preclinical study provides evidence that multiparametric MRI/MRE can be used noninvasively to assess disease severity and monitor treatment response in ALD.

Regulatory T Cells and Their Derived Cytokine, Interleukin-35, Reduce Pain in Experimental Autoimmune Encephalomyelitis.

Sensory problems such as neuropathic pain are common and debilitating symptoms in multiple sclerosis (MS), an autoimmune inflammatory disorder of the CNS. Regulatory T (Treg) cells are critical for maintaining immune homeostasis, but their role in MS-associated pain remains unknown. Here, we demonstrate that Treg cell ablation is sufficient to trigger experimental autoimmune encephalomyelitis (EAE) and facial allodynia in immunized female mice. In EAE-induced female mice, adoptive transfer of Treg cells and spinal delivery of the Treg cell cytokine interleukin-35 (IL-35) significantly reduced facial stimulus-evoked pain and spontaneous pain independent of disease severity and increased myelination of the facial nociceptive pathway. The effects of intrathecal IL-35 therapy were Treg-cell dependent and associated with upregulated IL-10 expression in CNS-infiltrating lymphocytes and reduced monocyte infiltration in the trigeminal afferent pathway. We present evidence for a beneficial role of Treg cells and IL-35 in attenuating pain associated with EAE independently of motor symptoms by decreasing neuroinflammation and increasing myelination.SIGNIFICANCE STATEMENT Pain is a highly prevalent symptom affecting the majority of multiple sclerosis (MS) patients and dramatically affects overall health-related quality of life; however, this is a research area that has been largely ignored. Here, we identify for the first time a role for regulatory T (Treg) cells and interleukin-35 (IL-35) in suppressing facial allodynia and facial grimacing in animals with experimental autoimmune encephalomyelitis (EAE). We demonstrate that spinal delivery of Treg cells and IL-35 reduces pain associated with EAE by decreasing neuroinflammation and increasing myelination independently of motor symptoms. These findings increase our understanding of the mechanisms underlying pain in EAE and suggest potential treatment strategies for pain relief in MS.

Interleukin-22 ameliorates acute-on-chronic liver failure by reprogramming impaired regeneration pathways in mice.

BACKGROUND & AIMS: Acute-on-chronic liver failure (ACLF) is a clinical syndrome defined by liver failure on pre-existing chronic liver disease. It is often associated with bacterial infection and high short-term mortality. Experimental models that fully reproduce ACLF are lacking, so too are effective pharmacological therapies for this condition. METHODS: To mimic ACLF conditions, we developed a severe liver injury model by combining chronic injury (chronic carbon tetrachloride [CCl4] injection), acute hepatic insult (injection of a double dose of CCl4), and bacterial infection (intraperitoneal injection of bacteria). Serum and liver samples from patients with ACLF or acute drug-induced liver injury (DILI) were used. Liver injury and regeneration were assessed to ascertain the potential benefits of interleukin-22 (IL-22Fc) administration. RESULTS: This severe liver injury model recapitulated some of the key features of clinical ACLF, including acute-on-chronic liver injury, bacterial infection, multi-organ injury, and high mortality. Liver regeneration in this model was severely impaired because of a shift from the activation of the pro-regenerative IL-6/STAT3 pathway to the anti-regenerative IFN-γ/STAT1 pathway. The impaired IL-6/STAT3 activation was due to the inability of Kupffer cells to produce IL-6; whereas the enhanced STAT1 activation was due to a strong innate immune response and subsequent production of IFN-γ. Compared to patients with DILI, patients with ACLF had higher levels of IFN-γ but lower liver regeneration. IL-22Fc treatment improved survival in ACLF mice by reversing the STAT1/STAT3 pathway imbalance and enhancing expression of many antibacterial genes in a manner involving the anti-apoptotic protein BCL2. CONCLUSIONS: Acute-on-chronic liver injury or bacterial infection is associated with impaired liver regeneration due to a shift from a pro-regenerative to an anti-regenerative pathway. IL-22Fc therapy reverses this shift and attenuates bacterial infection, thus IL-22Fc may have therapeutic potential for ACLF treatment. LAY SUMMARY: A mouse model combining chronic liver injury, acute hepatic insult, and bacterial infection recapitulates some of the key features of acute-on-chronic liver failure (ACLF) in patients. Both fibrosis and bacterial infection contribute to the impaired regenerative capacity of the liver in patients with ACLF. Herein, we show that IL-22Fc therapy improves ACLF by reprogramming impaired regenerative pathways and attenuating bacterial infection. Thus, it may have therapeutic potential for patients with ACLF.

Interleukin-21 enhances the antibody avidity elicited by DNA prime and MVA boost vaccine.

Enhancement of the magnitude or affinity of protective antibodies (Abs) induced by vaccine adjuvant is highly desirable to prevent challenging pathogens such as HIV-1. IL-21 plays a crucial role in germinal center reactions during humoral immune responses. However, the effect of IL-21 as a vaccine adjuvant on the quantity and quality of antigen-specific Abs elicited by DNA prime and MVA boost vaccine, a commonly used vaccine strategy, remains unknown. To close this knowledge gap, female adult B6N mice were primed with DNA vaccine twice (days 0, 14, 100 µg, I.M.) and boosted with MVA vaccine (day 28, 2 × 107 pfu, I.M.) with or without an IL-21 DNA adjuvant (days 3, 17, 31, 40 µg, I.M.), in which HIV-1 gag was expressed as a model antigen. With the addition of an IL-21 adjuvant, we found significantly increased avidity of antigen-specific Abs at multiple time points in a longitudinal follow up. Collectively, our results suggest that an IL-21 immune adjuvant can significantly increase Ab quality induced by heterologous DNA-MVA prime-boost vaccine strategy.

Role of interleukin-24 in the tumor-suppressive effects of interferon-ß on melanoma.

BACKGROUND: Type 1 interferons (IFNs), including IFN-ß, are widely used in adjuvant therapy for patients who undergo surgery for malignant melanoma to inhibit recurrence and in-transit metastasis. The precise mechanisms underlying the tumor-suppressive effects of IFN-ß on melanoma are not yet completely understood. OBJECTIVE: The purpose was to reveal the mechanisms underlying the tumor-suppressive effects of IFN-ß via interleukin (IL)-24. METHODS: Genome-wide oligonucleotide microarray, quantitative real-time reverse transcription-polymerase chain reaction (PCR), enzyme-linked immunosorbent assay and western blotting assay were performed using four melanoma cell lines (A375, RPMI-7951, SK-MEL-5 and SK-MEL-1) treated with natural-type IFN-ß to assess the expression of IL-24. Proliferation assay was performed using these melanoma cells and IL-24 knock-down melanoma cells. RESULTS: Genome-wide microarray analysis detected candidate genes upregulated in IFN-ß-sensitive cells after treatment with IFN-ß. We focused on IL-24 among the candidate genes encoding secretory proteins. Peak IL-24 mRNA expression completely correlated with the order of sensitivity of melanoma cells to IFN-ß. IFN-ß treatment induced extracellular IL-24 protein in IFN-ß-sensitive cells, but did not induce intracellular IL-24 protein. Knock-down of IL-24 changed melanoma cells into IFN-ß-resistant cells. The expression ratio of IL-22R1, one of the IL-24 receptors, correlated with the order of sensitivity of melanoma cells to IFN-ß. Treatment with recombinant human IL-24 did not have any effects on all the melanoma cell lines. CONCLUSION: Our data suggest that IFN-ß suppresses the proliferation of melanoma cells through extracellular IL-24 protein derived from melanoma cells.

IL-21 alleviates allergic asthma in DOCK8-knockout mice.

Patients with DOCK8 deficiency are at increased susceptibility to develop allergic diseases such as food allergy and asthma. Here, we aimed to analyze the pathogenesis of asthma in DOCK8-deficient patients. In our mouse model, DOCK8-knockout (KO) mice sensitized with low-dose OVA were challenged with 1.5% OVA to induce allergic asthma. As compared to that in WT mice, remarkable airway hyperresponsiveness was observed in KO mice. Increased inflammatory cells and eosinophils infiltrated in airway lumen in KO mice especially around bronchi. KO mice showed higher levels of serum IgE and OVA-specific IgE and significantly elevated IgE-producing B cells in blood and in spleen. Surprisingly, nasal administration with rmIL-21 significantly reduced the airway hyperresponsiveness, inflammatory infiltration, as well as the serum IgE and IgE-producing B cells. DOCK8-knockout mice are susceptible to low-dose OVA induced allergic airway inflammation and airway hyperresponsiveness. Supplementary nasal administration of rmIL-21 alleviates allergic asthma in this mouse model.

Oral Escherichia coli expressing IL-35 meliorates experimental colitis in mice.

BACKGROUND: Ulcerative colitis (UC) is a type of inflammatory bowel disease (IBD) characterized by chronic inflammation of colon. It is commonly believed that the imbalance of immune system and overwhelming production of cytokines are involved in the pathogenesis of UC. Recent studies demonstrated that interleukin-35 (IL-35), a key player in the regulation of inflammation, has been identified as potential therapeutic target to treat UC. However, conventional intravenous administration is costly and inconvenient. The present study was designed to establish a novel IL-35 delivery system and investigate its therapeutic effects on dextran sulfate sodium (DSS)-induced experimental colitis in mice for the first time. METHODS: An engineered Escherichia coli (E. coli/IL-35) expressing IL-35 was constructed. Adult male BALB/c mice randomly got the oral administration of E. coli/IL-35, empty plasmid-transformed E. coli (E. coli0) or PBS for treatment following ingestion of 3% DSS solution for 5 days. Normal mice were used as control group. Colonic and splenic tissues were collected on day 10 post-DSS-induction. Clinical signs, disease activity index (DAI), pathological and immunohistological changes, cytokine profiles and cell populations were evaluated. RESULTS: Intragastric administration of E. coli/IL-35 effectively protected the colitis mice from DSS assimilation including weight loss and colon shortening. Pathological analysis showed significantly lower DAI score and much less intra-colon infiltration of neutrophils and CD3+ cells in the IL-35 treated group. Moreover, E. coli/IL-35-treated mice demonstrated much less CD4+ IL-17A+ Th17 cells and a higher level of CD4+CD25+Foxp3+ Tregs in spleen and mesenteric lymph nodes, as well as increased colon and serum level of IL-10 and IL-35 and decreased levels of IL-6. CONCLUSIONS: Our study showed that E. coli/IL-35 as a novel oral IL-35 delivery system alleviated inflammatory damage of colonic tissue in the colitic mice. Genetic therapeutic strategies using engineered E. coli encoding immunoregulatory cytokines may provide a potential approach for the treatment of IBD.

IL-35 is a Protective Immunomodulator in Brain Ischemic Injury in Mice.

IL-35 has been identified as a novel anti-inflammatory cytokine that belongs to the IL-12 cytokine family and has been verified to play a protective role in autoimmune diseases. In this study, we investigated the protective effects of IL-35 on cerebral ischemia/reperfusion (I/R) injury in a middle cerebral artery occlusion mouse model. We determined that the expression of IL-35 was initially decreased and subsequently increased in I/R injury. Moreover, IL-35 (i.c.v.) pre- and posttreatment significantly reduced the infarct volume and improved neurological deficits after 45 min of ischemia and 24 h of reperfusion. Importantly, IL-35 treatment improved neurological function recovery, particularly in balance ability, at 14 days after treatment. Finally, our results showed that IL-35 treatment reduced the expression of IL-6 and IL-1ß, which are confirmed proinflammatory cytokines, thus indicating that these cytokines have both been linked to the anti-inflammatory mechanisms of IL-35. Therefore, IL-35 may be a key immune mediator in brain ischemic injury and appears to have promising potential for clinical trials.

Interferon lambda1/IL-29 and inorganic polyphosphate are novel regulators of neutrophil-driven thromboinflammation.

Neutrophils and neutrophil-released meshwork structures termed neutrophil extracellular traps (NETs) are major mediators of thromboinflammation and emerging targets for therapy, yet the mechanisms and pathways that control the role of neutrophils in thromboinflammation remain poorly understood. Here, we explored the role of IFN-λ1/IL-29, a major antiviral cytokine recently shown to suppress the neutrophil migratory capacity, in prothrombotic and proNETotic functions of neutrophils. In an ex vivo human experimental setting of acute ST-segment elevation myocardial infarction (STEMI), we show that IFN-λ1/IL-29 hinders NET release and diminishes the amount of cytoplasmic TF in neutrophils. Since platelet-neutrophil interaction plays a major role in NET-induced thromboinflammation, we further studied how IFN-λ1/IL-29 may interrupt this interaction. In this context, we identified inorganic polyphosphate (polyP) as a platelet-derived NET inducer in STEMI. In arterial STEMI thrombi, polyP was present in platelets and in close proximity to NET remnants. PolyP release from activated platelets was dependent on thrombin present in infarcted artery plasma, resulting in NET formation by promoting mTOR inhibition and autophagy induction. The effect of polyP on mTOR inhibition was counteracted by IFN-λ1/IL-29 treatment, leading to inhibition of NET formation. Consistently, we show in an in vivo model of FeCl3 -induced arterial thrombosis that IFN-λ2/IL-28A exerts strong antithrombotic potential. Taken together, these findings reveal a novel function of IFN-λ1/IL-29 in the suppression of thromboinflammation. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

CTL-Promoting Effects of IL-21 Counteract Murine Lupus in the Parent→F1 Graft-versus-Host Disease Model.

IL-21 promotes B cell and CTL responses in vivo, conferring IL-21 with a role in both humoral and cellular responses. Because CTL can target and eliminate autoreactive B cells, we investigated whether IL-21R signaling in CD8 T cells would alter the expansion of autoreactive B cells in an autoimmune setting. We addressed this question using the parent→F1 murine model of acute and chronic (lupus-like) graft-versus-host disease (GVHD) as models of a CTL-mediated or T-dependent B cell-mediated response, respectively. Induction of acute GVHD using IL-21R-deficient donor T cells resulted in decreased peak donor CD8 T cell numbers and decreased CTL effector function due to impaired granzyme B/perforin and Fas/Fas ligand pathways and a phenotype of low-intensity chronic GVHD with persistent host B cells, autoantibody production, and mild lupus-like renal disease. CTL effector maturation was critically dependent on IL-21R signaling in Ag-specific donor CD8, but not CD4, T cells. Conversely, treatment of DBA/2J→F1 chronic GVHD mice with IL-21 strongly promoted donor CD8 T cell expansion and rescued defective donor anti-host CTLs, resulting in host B cell elimination, decreased autoantibody levels, and attenuated renal disease, despite evidence of concurrently enhanced CD4 help for B cells and heightened B cell activation. These results demonstrate that, in the setting of lupus-like CD4 T cell-driven B cell hyperactivity, IL-21 signaling on Ag-specific donor CD8 T cells is critical for CTL effector maturation, whereas a lack of IL-21R downregulates CTL responses that would otherwise limit B cell hyperactivity and autoantibody production.

Critical Role of IL-22/IL22-RA1 Signaling in Pneumococcal Pneumonia.

IL-22-IL-22R signaling plays a crucial role in regulating host defenses against extracellular pathogens, particularly in the intestine, through the induction of antimicrobial peptides and chemotactic genes. However, the role of IL-22-IL-22R is understudied in Streptococcus pneumoniae lung infection, a prevalent pathogen of pneumonia. This paper presents the findings of IL-22 signaling during a murine model of pneumococcal pneumonia and improvement of bacterial burden upon IL-22 administration. IL-22 was rapidly induced in the lung during pneumococcal infection in wild-type mice, and Il22(-/-) mice had higher pneumococcal burdens compared with controls. Additionally, mice with hepatic-specific deletion of Il22ra1 also had higher bacterial burdens in lungs compared with littermate controls after intrapulmonary pneumococcal infection, suggesting that IL-22 signaling in the liver is important to control pneumococcal pneumonia. Thus, we hypothesized that enhancement of IL-22 signaling would control pneumococcal burden in lung tissues in an experimental pneumonia model. Administration of rIL-22 systemically to infected wild-type mice decreased bacterial burden in lung and liver at 24 h postinfection. Our in vitro studies also showed that mice treated with IL-22 had increased C3 expression in the liver compared with the isotype control group. Furthermore, serum from mice treated with IL-22 had improved opsonic capacity by increasing C3 binding on S. pneumoniae Taken together, endogenous IL-22 and hepatic IL-22R signaling play critical roles in controlling pneumococcal lung burden, and systemic IL-22 decreases bacterial burden in the lungs and peripheral organs by potentiating C3 opsonization on bacterial surfaces, through the increase of hepatic C3 expression.

The Superiority of IFN-λ as a Therapeutic Candidate to Control Acute Influenza Viral Lung Infection.

Here, we studied the IFN-regulated innate immune response against influenza A virus (IAV) infection in the mouse lung and the therapeutic effect of IFN-λ2/3 in acute IAV lung infection. For viral infections, IAV (WS/33, H1N1, PR8 H1N1, H5N1) were inoculated into wild-type mice by intranasal delivery, and IAV mRNA level and viral titer were measured. To compare the antiviral effect of IFNs in vivo in the lung, neutralizing antibodies and recombinant IFNs were used. After intranasal inoculation of IAV into mice, viral infection peaked at 7 days postinfection, and the IAV titer also reached its peak at this time. We found that IFN-ß and IFN-λ2/3 were preferentially induced after IAV infection and the IFN-λ2/3-mediated innate immune response was specifically required for the induction of IFN-stimulated genes (ISGs) transcription in the mouse respiratory tract. Neutralization of secreted IFN-λ2/3 aggravated acute IAV lung infection in mice with intact IFN-ß induction; consistent with this finding, the transcription of ISGs was significantly reduced. Intranasal administration of IFN-λ2/3 significantly suppressed various strains of IAV infection, including WS/33 (H1N1), PR (H1N1), and H5N1 in the mouse lung, and was accompanied by greater up-regulation of ISGs. Taken together, our data indicate that the IFN-λ2/3-mediated innate immune response is necessary to protect the lungs from IAV infection, and intranasally delivered IFN-λ2/3 has the potential to be a useful therapeutic strategy for treating acute IAV lung infection.