STAT1 KO Mice Research Articles

91: Type I interferon-mediated host response against viral infection in the absence of STAT1

Canonical type I interferon (IFN-I) signaling through interferon stimulated gene factor (ISGF) 3 has a key role in host innate and adaptive immune responses during viral infection. We have previously found that following systemic infection with lymphocytic choriomeningitis virus (LCMV), STAT1 KO but not WT, IFNAR KO or STAT1/IFNAR DKO mice develop lethal disease characterized by a ‘cytokine storm’. Furthermore, CD4 + T cells were shown to be perpetrators of this lethal phenotype. This study aims to determine the underlying molecular mechanisms of this lethal disease. As an in vitro model of antigen presenting cells (APCs), bone marrow-derived macrophages (BMDMs) from WT, STAT1 KO, IFNAR KO and STAT1/IFNAR DKO were infected with LCMV. The induction of ISG15 mRNA was significantly higher in infected WT BMDMs compared with the other genotypes, consistent with the loss of a canonical anti-viral response in the absence of STAT1 and/or IFNAR. In contrast, infected STAT1 KO, IFNAR KO and STAT1/IFNAR DKO BMDMs showed comparably increased mRNA levels for various pro-inflammatory cytokines (e.g. IL-6 and IL-1 β ) . Furthermore, phosphorylation of STAT3, STAT6, NF κ B and ERK was increased in infected BMDMs independent of the genotype. However, pY-STAT2 induction was only observed in infected WT BMDMs. The activation of these signaling molecules was also observed in liver and kidney extracts from LCMV-infected WT, STAT1 KO, IFNAR KO and STAT1/IFNAR DKO mice. In particular, activation of STAT2 was found in WT and STAT1 KO mice only and pY-STAT2 levels were significantly higher in the absence of STAT1 during infection. These studies show that LCMV infection in BMDMs and in vivo induces: (1) a core host response that is independent of IFN-I and (2) a non-canonical STAT1-independent IFN-I-dependent signaling response.

Role of signal transducer and activator of transcription 6 in the development of colitis associated colorectal cancer (TUM7P.959)

Abstract STAT6 signaling pathway has important pathophysiologic implications in a variety of cell types and diseases. On cancer studies, mice lacking STAT6 display enhanced tumor immunity to both primary and metastatic mammary carcinomas. In humans, STAT6 protein has been found to be constitutively activated in several cancer types. However, the role of STAT6 in influencing the resistance or susceptibility of colon cancer cells to apoptosis and invasiveness/metastasis has not yet been validated during in vivo colitis-associated colon cancer (CAC).This study aims to identify the effects of STAT6 in tumorigenesis utilizing STAT6-KO mice in an experimental CAC model and evaluating changes in immune cell populations in the absence of this gene. CAC was induced in both STAT6-KO and WT mice by injection of 12.5 mg/kg AOM followed by three rounds of 2% DSS exposure to elicit colitis. On day 63 after CAC induction, mice were sacrificed. Colonic inflammation, proliferation and tumorigenesis were evaluated. Tumor numbers per mouse (0.8 versus 19.6; P=0.0001), and bowel weight (0.3 versus 0.6 g; P=0.05) were significantly decreased in STAT6-KO mice compared to WT mice. Furthermore, both inflammation and proliferation scores in colon from STAT6-KO mice were significantly lower than WT mice. Although additional studies are needed to clarify the mechanism(s) underlying improved anti-tumor immunity in STAT6-KO mice, these results may be a basis for an immunotherapy strategy in CAC development.

A suppressive role of STAT1 in lupus-like chronic graft versus host disease (IRM7P.479)

Abstract Systemic lupus erythematosus (SLE) is a multisystem autoimmune disease driven by genetic and environmental factors with unknown etiopathogenesis. The bm12→B6 chronic graft-versus-host disease (cGVHD) is a well-studied inducible mouse model of SLE. STAT1 is dynamically regulated during the course of immune and inflammatory responses. However, little is known about the role of STAT1 in autoimmune diseases. We induced cGVHD in B6.STAT1-KO and B6.WT mice by intraperitoneally injection of 1×108 splenocytes from bm12 mice. Mice were followed for autoimmune manifestations, such as mortality, skin rash, and renal involvement. Upon injection, WT mice developed anti-dsDNA autoantibodies starting at week 2 (as expected), with decline noted after week 4. In contrast, STAT1-deficient mice exhibited a prolonged and significant increase of anti-dsDNA autoantibody responses compared to WT mice (week 4 to week 8). Severe splenomegaly also developed in cGVHD STAT1-KO mice. Increased autoantibody titers were accompanied by increased proteinuria in mice lacking STAT1 due to immune complex deposition. Three months after injection, 40% of the STAT1-KO mice with cGVH disease were dead. Anti-dsDNA isotypes in cGVH diseased STAT1-KO mice were enhanced (IgG1 responses), but IgG3 responses were absent. STAT1 seems to suppress systemic autoimmunity by altering autoantibody isotype switching. Results from these studies may provide insights into the pathogenesis of STAT1 in SLE.

Type-2 innate lymphoid cells facilitate antigen-induced CD4+ TH2 cell immune response that exacerbates chronic allergic asthma (HYP7P.291)

Abstract Type-2 innate lymphoid cells (ILC2s) and acquired CD4+TH2 and TH17 cells were shown to contribute to the pathogenesis of allergic asthma; however, their relative roles in mounting antigen recall response that exacerbate chronic allergic asthma remain elusive. Here we reported that repeated OVA re-challenge induced significant increases of eosinophil, not neutrophil accumulation and serum OVA specific IgE and IgG1 in rested mice that previously developed allergic asthma. Subsequent analysis revealed that these rested mice with allergic asthma preferentially mounted elevated OVA specific CD4+TH2 cell response against intranasal OVA re-challenge, while the frequency of lung OVA specific TH17 cells or ILC2s remained constant. While ILC2s and TH17 cells developed normally, STAT6 KO mice that lacked TH2 cell compartment or mice ablated of CD4+ T cells failed to mount allergic recall response to OVA re-challenge. Notably, we observed that cocultured ILC2s could greatly enhance TH2 cytokine production by OVA specific TH2 cells after OVA stimulation ex vivo. Indeed, mice co-transferred with ILC2s and OVA specific TH2 cells exhibited significantly increased inflammatory cell infiltration and goblet cell hyperplasia, compared to those transferred with ILC2s or TH2 cells alone. Collectively, our results suggest that antigen re-exposures preferentially induce lung resident TH2 cells to mount antigen recall immune response that can be enhanced by ILC2s to exacerbate allergic inflammation

IL-4 Suppresses the Responses to TLR7 and TLR9 Stimulation and Increases the Permissiveness to Retroviral Infection of Murine Conventional Dendritic Cells

Th2-inducing pathological conditions such as parasitic diseases increase susceptibility to viral infections through yet unclear mechanisms. We have previously reported that IL-4, a pivotal Th2 cytokine, suppresses the response of murine bone-marrow-derived conventional dendritic cells (cDCs) and splenic DCs to Type I interferons (IFNs). Here, we analyzed cDC responses to TLR7 and TLR9 ligands, R848 and CpGs, respectively. We found that IL-4 suppressed the gene expression of IFNβ and IFN-responsive genes (IRGs) upon TLR7 and TLR9 stimulation. IL-4 also inhibited IFN-dependent MHC Class I expression and amplification of IFN signaling pathways triggered upon TLR stimulation, as indicated by the suppression of IRF7 and STAT2. Moreover, IL-4 suppressed TLR7- and TLR9-induced cDC production of pro-inflammatory cytokines such as TNFα, IL-12p70 and IL-6 by inhibiting IFN-dependent and NFκB-dependent responses. IL-4 similarly suppressed TLR responses in splenic DCs. IL-4 inhibition of IRGs and pro-inflammatory cytokine production upon TLR7 and TLR9 stimulation was STAT6-dependent, since DCs from STAT6-KO mice were resistant to the IL-4 suppression. Analysis of SOCS molecules (SOCS1, −2 and −3) showed that IL-4 induces SOCS1 and SOCS2 in a STAT6 dependent manner and suggest that IL-4 suppression could be mediated by SOCS molecules, in particular SOCS2. IL-4 also decreased the IFN response and increased permissiveness to viral infection of cDCs exposed to a HIV-based lentivirus. Our results indicate that IL-4 modulates and counteracts pro-inflammatory stimulation induced by TLR7 and TLR9 and it may negatively affect responses against viruses and intracellular parasites.

Signal transducer and activator of transcription-1 localizes to the mitochondria and modulates mitophagy

The signal transducer and activator of transcription (STAT) proteins are latent transcription factors that have been shown to be involved in cell proliferation, development, apoptosis, and autophagy. STAT proteins undergo activation by phosphorylation at tyrosine 701 and serine 727 where they translocate to the nucleus to regulate gene expression. STAT1 has been shown to be involved in promoting apoptotic cell death in response to cardiac ischemia/reperfusion and has recently been shown by our laboratory to be involved in negatively regulating autophagy. These processes are thought to promote cell death and restrict cell survival leading to the generation of an infarct. Here we present data that shows STAT1 localizes to the mitochondria and co-immunoprecipitates with LC3. Furthermore, electron microscopy studies also reveal mitochondria from ex vivo I/R treated hearts of STAT1KO mice contained within a double membrane autophagosome indicating that STAT1 may be involved in negatively regulating mitophagy. This is the first description of STAT1 being localized to the mitochondria and also having a role in mitophagy.

32: IRF7-dependent, type I IFN (IFN-I) production induces lethal immune-mediated disease in STAT1 KO mice infected with LCMV

STAT1 KO but not WT, STAT2 KO, IRF9 KO or IFNAR KO mice succumb to a lethal CD4+ T cell-mediated disease following systemic infection with lymphocytic choriomeningitis virus (LCMV). In previous studies we showed: (i) LCMV infection induces IRF7 gene expression in a STAT1-independent, but STAT2-dependent manner, and (2) IRF7 is required for IFN-alpha but not IFN-βeτα production in LCMV infection. Here we examined the role of IRF7 in the lethal host response to LCMV infection in STAT1 KO mice. In contrast to STAT1 KO mice, STAT1/IRF7 double KO mice survived LCMV infection and had a marked reduction in immune pathology affecting key organs including the liver, kidney and lung. LCMV infection in STAT1 KO mice was associated with a significant elevation of a number of cytokines in the serum including IFN-αλπηα, IFN-βeτα, IFN-γαμμα, IL-5, IL-6 and MCP1 but this response was absent in STAT1/IRF7 KO mice which had a modest increase in MCP-1 only. In addition, in the spleen of LCMV-infected STAT1 KO mice, IFN-βeτα and IFN-γαμμα mRNAs were also much more highly induced when compared with WT mice, while in STAT1/IRF7 KO mice IFN-γαμμα mRNA induction was similar to WT animals, there was no induction of IFN-βeτα mRNA. To elucidate further a possible role of IFN-I in lethal disease, we examined STAT1/IFNAR double KO mice. In contrast to STAT1 KO mice that all succumbed to lethal disease, STAT1/IFNAR KO mice all survived infection with LCMV. In conclusion, lethality resulting from LCMV infection in STAT1 KO mice requires IRF7-dependent, IFN-I production which drives a catastrophic antiviral immune response.

227: IL-27 affects helper T cell response via regulation of PGE2 production by macrophages

IL-27 is a heterodimeric cytokine that regulates both innate and adaptive immunity. Although IL-27 was initially reported as a Th1 inducer by the induction of IL-12R on naive CD4+T cells, recently it is revealed that IL-27 acts as an immune suppressor by the induction of Tr1 type T cells, which produce IL-10 and inhibit effector T cells. IL-27 also suppresses function of macrophages and dendritic cells by regulation of cytokine secretion. On the other hand, effects of IL-27 on regulation of inflammation via mediators other than cytokines remain poorly understood. To address this issue, we examined immunoregulatory effects of conditional medium of bone marrow-derived macrophages (BMDMs) from WSX-1 (IL-27Ra) KO mice to find enhanced IFN-g and IL-17A secretion by CD4+T cells as compared with that of control BMDMs. This enhancement was abolished by EP2 and EP4 antagonist, suggesting possible involvement of prostaglandins. We then found the PGE2 secretion and COX-2 expression by BMDMs from WSX-1 KO mice was increased compared to control macrophages in response to LPS. To explore how IL-27/WSX-1 signal regulate COX-2/PGE2 production, murine WSX-1-expressing Raw264.7 cells (mWSX-1-Raw264.7) were established. mWSX-1-Raw264.7 showed phosphorylation of both STAT1 and STAT3 in response to IL-27 and produced less amounts of PGE2 and COX-2 compared to parental Raw264.7. Finally, to examine whether STAT1 and STAT3 downstream of IL-27R regulate PGE2 and COX-2 production, siRNA knockdown study was performed and siSTAT1, but not siSTAT3, resulted in increased production of PGE2 and COX-2. In addition, BMDMs from STAT1 KO mice showed higher COX-2 expression than those from control mice. Collectively, our result indicated that IL-27/WSX-1 regulated PGE2 secretion via STAT1-COX-2 pathway in macrophages and affected helper T cells response in a PGE2-mediated indirect fashion.

The role of innate versus adaptive immune responses in a mouse model of O'nyong-nyong virus infection.

O'nyong-nyong virus (ONNV), an alphavirus closely related to chikungunya virus (CHIKV), has caused three major epidemics in Africa since 1959. Both ONNV and CHIKV produce similar syndromes with fever, rash, and debilitating arthralgia. To determine the roles of the innate and adaptive immune responses, we infected different knockout mice with two strains of ONNV (SG650 and MP30). Wild-type, RAG1 KO, and IFNγR KO mice showed no signs of illness or viremia. The STAT1 KO and A129 mice exhibited 50-55% mortality when infected with SG650. Strain SG650 was more virulent in the STAT1 KO and A129 than MP30. Deficiency in interferon α/β signaling (A129 and STAT1 KO) leaves mice susceptible to lethal disease; whereas a deficiency of interferon γ signaling alone had no effect on survival. Our findings highlight the importance of type I interferon in protection against ONNV infection, whereas the adaptive immune system is relatively unimportant in the acute infection.

Deacetylation of C/EBPβ is required for IL‐4‐induced arginase‐1 expression in murine macrophages

The amount of arginine available at inflammatory loci is a limiting factor for the growth of several cells of the immune system. IL-4-induced activation of macrophages produced arginase-1, which converts arginine into ornithine, a precursor of polyamines and proline. Trichostatin A (TSA), a pan-inhibitor of histone deacetylases (HDACs), inhibited IL-4-induced arginase-1 expression. TSA showed promoter-specific effects on the IL-4-responsive genes. While TSA inhibited the expression of arginase-1, fizz1, and mrc1, other genes, such as ym,1 mgl1, and mgl2, were not affected. The inhibition of arginase-1 occurred at the transcriptional level with the inhibition of polymerase II binding to the promoter. IL-4 induced STAT6 phosphorylation and binding to DNA. These activities were not affected by TSA treatment. However, TSA inhibited C/EBPβ DNA binding. This inhibitor induced acetylation on lysine residues 215-216, which are critical for DNA binding. Finally, using macrophages from STAT6 KO mice we showed that STAT6 is required for the DNA binding of C/EBPβ. These results demonstrate that the acetylation/deacetylation balance strongly influences the expression of arginase-1, a gene of alternative activation of macrophages. These findings also provide a molecular mechanism to explain the control of gene expression through deacetylase activity.

Analysis of T and B cell phenotype and effector memory T cell response in a mouse model of tree nut allergy (125.10)

Abstract Tree nuts cause potentially fatal anaphylactic reactions for reasons that are not well understood. We previously reported an adjuvant free mouse model of hazelnut allergy. Here we analyzed the T & B cell phenotype and effector memory responses. Balb/c mice were transdermally sensitized with hazelnut. Hazelnut allergy was confirmed by measuring hazelnut specific IgE antibodies and systemic anaphylaxis (hypothermia) upon challenge. Using flow cytometry T cell and B cell phenotypes were studied. Hazelnut allergic mice exhibited a significant increase in the number of CD4+ CD62L - T cells in the bone marrow and the spleen. Among B cells, there was increase in CD80+ cells in BM, spleen and MLN and CD62L- cells in BM and spleen of allergic mice. Among CD80+ B cells a significant proportion were CD73- in the MLN of allergic mice. Effector memory Type-2 cytokine responses were analyzed using spleen cell cultures. Allergic mice exhibited a robust IL-4, IL-5 and IL-13 response that was blocked by anti-CD4 mAb. The STAT6 KO mice exhibited IL-5 and IL-13 but not IL-4 memory response. These data demonstrate that hazelnut elicits: (i) significant expansion of CD4+CD62L- T cells in both the BM and the periphery; (ii) a modest increase in CD80+ and CD62L- B cells in BM and the periphery; and a significant down regulation of CD73 expression on a subset of CD80+ B cells in the MLN; and (iii) STAT6 and CD4 dependent IL-4 memory; and STAT6 independent, CD4 dependent IL-5/IL-13 memory.

Mice Deficient in STAT1 but Not STAT2 or IRF9 Develop a Lethal CD4+T-Cell-Mediated Disease following Infection with Lymphocytic Choriomeningitis Virus

Interferon (IFN) signaling is crucial for antiviral immunity. While type I IFN signaling is mediated by STAT1, STAT2, and IRF9, type II IFN signaling requires only STAT1. Here, we studied the roles of these signaling factors in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild-type (WT) mice and mice lacking either STAT2 or IRF9, LCMV infection was nonlethal, and the virus either was cleared (WT) or established persistence (STAT2 knockout [KO] and IRF9 KO). However, in the case of STAT1 KO mice, LCMV infection was lethal and accompanied by severe multiorgan immune pathology, elevated expression of various cytokine genes in tissues, and cytokines in the serum. This lethal phenotype was unaltered by the coabsence of the gamma interferon (IFN-γ) receptor and hence was not dependent on IFN-γ. Equally, the disease was not due to a combined defect in type I and type II IFN signaling, as IRF9 KO mice lacking the IFN-γ receptor survived infection with LCMV. Clearance of LCMV is mediated normally by CD8(+) T cells. However, the depletion of these cells in LCMV-infected STAT1 KO mice was delayed, but did not prevent, lethality. In contrast, depletion of CD4(+) T cells prevented lethality in LCMV-infected STAT1 KO mice and was associated with a reduction in tissue immune pathology. These studies highlight a fundamental difference in the role of STAT1 versus STAT2 and IRF9. While all three factors are required to limit viral replication and spread, only STAT1 has the unique function of preventing the emergence of a lethal antiviral CD4(+) T-cell response.

Community Dynamics in the Mouse Gut Microbiota: A Possible Role for IRF9-Regulated Genes in Community Homeostasis

BackgroundGut microbial communities of mammals are thought to show stable differences between individuals. This means that the properties imparted by the gut microbiota become a unique and constant characteristic of the host. Manipulation of the microbiota has been proposed as a useful tool in health care, but a greater understanding of mechanisms which lead to community stability is required. Here we have examined the impact of host immunoregulatory phenotype on community dynamics.Methods and FindingsDenaturing gradient gel electrophoresis was used to analyse the faecal bacterial community of BALB/c and C57BL/6 mice and C57BL/6 mice deficient for either type I interferon (IFN) signalling (IRF9 KO mice) or type I and type II IFN signalling (STAT1 KO mice). Temporal variation was found in all mouse strains. A measure of the ability for a community structure characteristic of the host to be maintained over time, the individuality index, varied between mouse strains and available data from pigs and human models. IRF9 KO mice had significantly higher temporal variation, and lower individuality, than other mouse strains. Examination of the intestinal mucosa of the IRF9 KO mice revealed an increased presence of T-cells and neutrophils in the absence of inflammation.SignificanceThe high temporal variation observed in the gut microbiota of inbred laboratory mice has implications for their use as experimental models for the human gut microbiota. The distinct IRF9 and STAT1 phenotypes suggest a role for IRF9 in immune regulation within the gut mucosa and that further study of interferon responsive genes is necessary to understand host-gut microbe relationships.

Staphylococcus aureus Biofilm-Mediated Infections: Characterization of the Host Adaptive Immune Response and its Role in Chronic Infection (45.2)

Abstract Staphylococcus aureus is a common cause of prosthetic implant infections, which can become chronic due to the ability of S. aureus to grow as a biofilm. Little is known about adaptive immune responses to these infections in vivo. We hypothesized that S. aureus elicits inflammatory Th1/Th17 responses, associated with biofilm formation, instead of protective Th2/Treg responses. A previously developed mouse model of prosthetic implant infection was modified to produce a long term biofilm infection and used to determine chronic infection rates, Treg frequency, and local cytokine levels in B6 and BALB/c mice. Chronic infection levels in STAT6 KO mice (BALB/c background), BALB/c mice receiving anti-CD25 MAb, and B6 mice receiving anti-IL-6 or anti-IL-12 p40 MAb were also assessed. Local S. aureus implant infection was chronic in 100% of B6 mice, whereas BALB/c mice more effectively cleared the infection and demonstrated higher levels of local Th2 cytokines and Tregs. STAT6 KO mice lost the ability to effectively clear an S. aureus implant infection, as did BALB/c mice receiving anti-CD25. In contrast, B6 mice treated with anti-IL-12 p40 were able to more effectively clear the infection. These results indicate that Th2/Treg responses are mechanisms of protection against chronic S. aureus implant infection, as opposed to Th1/Th17 responses, which may play a role in development of chronic implant infection. Our results also suggest that anti-IL-12 p40 MAb may be used therapeutically.

Crucial Role of Interferon-γ in Experimental Autoimmune Prostatitis

An autoimmune etiology is proposed in some patients with chronic nonbacterial prostatitis since they show interferon-gamma secreting lymphocytes specific to prostate antigens in the periphery and increased interferon-gamma in seminal plasma. We investigated the involvement of interferon-gamma in an animal model of autoimmune prostatitis. Experimental autoimmune prostatitis was studied in the no-obese diabetic and C57Bl/6 (Harlan, Zeist, The Netherlands) susceptible mouse strains, and in the IRF-1 KO and STAT-1 KO mouse strains deficient in transcription factors involved in interferon-gamma signaling. Experimental autoimmune prostatitis was characterized by prostate specific interferon-gamma secreting cells in the periphery and by T-helper 1 related cytokines in the target organ. Increased interferon-gamma and interleukin-12 were observed in the prostate of autoimmune animals while interleukin-10 and interleukin-4 were decreased and unaltered, respectively. The absence of transcription factors involved in the interferon-gamma signaling cascade, IRF-1 and STAT-1, made mice resistant to experimental autoimmune prostatitis. IRF-1 KO and STAT-1 KO mice immunized with prostate antigens did not show infiltration or alterations in the prostate. They did not have the typical prostate specific autoimmune response and showed decreased interferon-gamma, interleukin-12 and interleukin-10, and augmented interleukin-4 in the prostate. Our results argue for a crucial role of interferon-gamma as a key factor in the pathogenesis of the disease. Intense research is promptly required to identify the pathogenic mechanisms underlying chronic prostatitis/chronic pelvic pain syndrome to find a more rational therapy.

Functional recovery and facial motoneuron survival are influenced by immunodeficiency in crush-axotomized mice

Facial nerve axotomy is a well-described injury paradigm for peripheral nerve regeneration and facial motoneuron (FMN) survival. We have previously shown that CD4 + T helper (Th) 1 and 2 effector subsets develop in the draining cervical lymph node, and that the IL-4/STAT-6 pathway of Th2 development is critical for FMN survival after transection axotomy. In addition, delayed behavioral recovery time in immunodeficient mice may be due to the absence of T and B cells. This study utilized a crush axotomy paradigm to evaluate FMN survival and functional recovery in WT, STAT-6 KO (impaired Th2 response), T-Bet KO (impaired Th1 response), and RAG-2 KO (lacking mature T and B cells) mice to elucidate the contributions of specific CD4 + T cell subsets in motoneuron survival and recovery mechanisms. STAT-6 KO and RAG-2 KO mice exhibited decreased FMN survival after crush axotomy compared to WT, supporting a critical role for the Th2 effector cell in motoneuron survival before target reconnection. Long term FMN survival was sustained through 10 wpo after crush axotomy in both WT and RAG-2 KO mice, indicating that target derived neurotrophic support maintains FMN survival after target reconnection. In addition, RAG-2 KO mice exhibited delayed functional recovery compared to WT mice. Both STAT-6 and T-Bet KO mice exhibited partially delayed functional recovery compared to WT, though not to the extent of RAG-2 KO mice. Collectively, our findings indicate that both pro- and anti-inflammatory CD4 + T cell responses contribute to optimal functional recovery from axotomy-induced facial paralysis, while FMN survival is supported by the anti-inflammatory Th2 response alone.

Lethal viral infection results from STAT1 but not STAT2 or IRF9 deficiency in mice and is mediated by CD4+ T-cells (44.11)

Abstract Type I IFN (α/β) signaling is crucial in the antiviral response and is mediated by STAT1, STAT2 and IRF9 while type II IFN (γ) uses STAT1. We studied the role of STAT1, STAT2 and IRF9 in the host response to systemic infection with lymphocytic choriomeningitis virus (LCMV). In wild type (WT), STAT2 KO and IRF9 KO mice, infection was non-lethal and virus was either cleared (WT) or established persistence (STAT2 and IRF9 KO). In contrast, in STAT1 KO mice, infection was lethal with severe immune pathology with T-cells, macrophages and granulocytes and elevated expression of various cytokines, particularly IFN-γ, present in various organs. However, lethality was unaltered in LCMV-infected STAT1 KO mice that lacked the IFN-γ receptor. While clearance of LCMV in WT mice is mediated by CD8+ T-cells, depletion of these cells in LCMV-infected STAT1 KO mice did not affect lethality. However, depletion of CD4+ T-cells prevented lethality in LCMV-infected STAT1 KO mice with a considerable reduction in tissue immune pathology. In vitro stimulation assays revealed a skewing of the T-cell response to the Th17 phenotype in LCMV-infected STAT1 KO but not WT mice. These findings indicated that STAT1 has a major protective function in LCMV infection, not only being crucial for limiting viral replication and spread, but also preventing the emergence of a lethal CD4+ T-cell response possibly mediated by Th17 cells. Support : NHMRC grant 512407.

Effects of immunodeficiency on motoneuron survival and peripheral nerve regeneration following facial nerve axotomy

Facial nerve axotomy is a well‐described injury model for peripheral nerve regeneration and facial motoneuron (FMN) survival. After facial nerve transection, FMN survival levels in Rag2 KO mice (lacking functional T and B cells) are ~60% compared to ~86% in wild‐type (WT) mice at 4 week postoperative (wpo). This decrease in Rag2 KO FMN survival can be rescued to WT levels by reconstitution with CD4+T, but not CD8+ or B cells, prior to nerve transection. However, the ability of CD4+T cells to support FMN survival appears to be transient because WT FMN survival levels begin to decrease by 10 wpo, approaching Rag2 KO levels. The first goal of this study is to examine WT FMN survival at extended time points (18 and 26 wpo) to determine whether a functional immune system provides transient neuroprotection, or long‐term support for FMN survival. In addition, we will utilize a crush axotomy to evaluate functional recovery in WT, STAT6 KO (impaired Th2 response), T‐Bet KO (impaired Th1 response), and Rag2 KO mice. Based on the immune‐mediated neuroprotection observed following transection, we speculate that some or all of these immunodeficient mouse models will have impaired functional recovery and/or decreased FMN survival after crush. The specific transgenic models used in this study may also confirm the role of CD4+T cell subsets in immune‐mediated neuroprotection.Supported by NIH grant NS40433 (KJJ & VMS).Grant Funding SourceNIH grant NS40433

Role of Interferon in Homologous and Heterologous Rotavirus Infection in the Intestines and Extraintestinal Organs of Suckling Mice

Recent studies demonstrated that viremia and extraintestinal rotavirus infection are common in acutely infected humans and animals, while systemic diseases appear to be rare. Intraperitoneal infection of newborn mice with rhesus rotavirus (RRV) results in biliary atresia (BA), and this condition is influenced by the host interferon response. We studied orally inoculated 5-day-old suckling mice that were deficient in interferon (IFN) signaling to evaluate the role of interferon on the outcome of local and systemic infection after enteric inoculation. We found that systemic replication of RRV, but not murine rotavirus strain EC, was greatly enhanced in IFN-alpha/beta and IFN-gamma receptor double-knockout (KO) or STAT1 KO mice but not in mice deficient in B- or T-cell immunity. The enhanced replication of RRV was associated with a lethal hepatitis, pancreatitis, and BA, while no systemic disease was observed in strain EC-infected interferon-deficient mice. In IFN-alpha/beta receptor KO mice the extraintestinal infection and systemic disease were only moderately increased, while RRV infection was not augmented and systemic disease was not present in IFN-gamma receptor KO mice. The increase of systemic infection in IFN-deficient mice was also observed during simian strain SA11 infection but not following bovine NCDV, porcine OSU, or murine strain EW infection. Our data indicate that the requirements for the interferon system to inhibit intestinal and extraintestinal viral replication in suckling mice vary among different heterologous and homologous rotavirus strains, and this variation is associated with lethal systemic disease.

The Role of TRAF2 Binding to the Type I Interferon Receptor in Alternative NFκB Activation and Antiviral Response

Type I interferons (IFNs) play critical roles in the host defense by modulating gene expression through the IFN-dependent activation of STAT and NFkappaB transcription factors. Previous studies established that IFN activates NFkappaB through a classical NFkappaB pathway that results in IkappaBalpha degradation and formation of p50-containing NFkappaB complexes, as well as an alternative pathway that involves NFkappaB-inducing kinase and TRAF2, which results in the formation of p52-containing NFkappaB complexes. In this study, we examined the interaction of TRAF proteins with the type I IFN receptor. We found that TRAF2 was directly coupled to the signal-transducing IFNAR1 subunit of the IFN receptor. By immunoprecipitation, overexpression of epitope-tagged IFNAR1 constructs, and glutathione S-transferase pulldown experiments, we demonstrate that TRAF2 rapidly binds to the IFNAR1 subunit of the IFN receptor upon IFN binding. The membrane proximal half of the IFNAR1 subunit was found to directly bind TRAF2. Moreover, analysis of mouse embryo fibroblasts derived from TRAF2 knock-out mice demonstrated that TRAF2 plays a critical role in the activation of the alternative NFkappaB pathway by IFN, but not the classical NFkappaB pathway, as well as in the antiviral action of IFN. Our results place TRAF2 directly in the signaling pathway transduced through the IFNAR1 subunit of the IFN receptor. These findings provide an important insight into the molecular mechanisms by which IFN generates signals to induce its biological effects.