Induce IFN Production Research Articles

In Silico Design of CT26 Polytope and its Surface Display by ClearColi™-Derived Outer Membrane Vesicles as a Cancer Vaccine Candidate Against Colon Carcinoma.

Simultaneous targeting of several mutations can be useful in colorectal cancer (CRC) due to its heterogeneity and presence of somatic mutations. As CT26 mutations and expression profiles resemble those of human CRC, we focused on designing a polyepitope vaccine based on CT26 neoepitopes. Due to its low immunogenicity, outer membrane vesicles (rOMV) as an antigen delivery system and adjuvant was applied. Herein, based on previous experimental and our in silico studies four CT26 neoepitopes with the ability to bind MHC-I and MHC-II, TCR, and induce IFN-α production were selected. To increase their immunogenicity, the gp70 and PADRE epitopes were added. The order of the neoepitopes was determined through 3D structure analysis using ProSA, Verify 3D, ERRAT, and Ramachandran servers. The stable peptide-protein docking between the selected epitopes and MHC alleles strengthen our prediction. The CT26 polytope vaccine sequence was fused to the C-terminal of cytolysin A (ClyA) anchor protein and rOMVs were isolated from endotoxin-free ClearColi™ strain. The results of the C-ImmSim server showed that the ClyA-CT26 polytope vaccine could induce T and B cells immunity.The ClyA-CT26 polytope was characterized as a soluble, stable, immunogen, and non-allergen vaccine and optimized for expression in ClearColi™ 24h after induction with 1mM IPTG at 25°C. Western blot analysis confirmed the expression of ClyA-CT26 polytope by ClearColi™ and also on ClearColi™-derived rOMVs. In conclusion, we found that ClearColi™-derived rOMVs with CT26 polytope can deliver CRC neoantigens and induce antitumor immunity, but in vivo immunological studies are needed to confirm vaccine efficacy.

Heyndrickxia coagulans strain SANK70258 suppresses symptoms of upper respiratory tract infection via immune modulation: a randomized, double-blind, placebo-controlled, parallel-group, comparative study.

Probiotic consumption strongly influences local intestinal immunity and systemic immune status. Heyndrickxia coagulans strain SANK70258 (HC) is a spore-forming lactic acid bacterium that has immunostimulatory properties on peripheral tissues. However, few reports have examined the detailed effectiveness of HC on human immune function and its mechanism of action. Therefore, we conducted a randomized, double-blind, placebo-controlled, parallel-group study to comprehensively evaluate the effects of HC on immunostimulatory capacity, upper respiratory tract infection (URTI) symptoms, and changes in intestinal organic-acid composition. Results of a questionnaire survey of URTI symptoms showed that runny nose, nasal congestion, sneezing, and sore throat scores as well as the cumulative number of days of these symptoms were significantly lower in the HC group than in the placebo group during the study period. Furthermore, the salivary secretory immunoglobulin A (sIgA) concentration was significantly higher, and the natural killer (NK) cell activity tended to be higher in the HC group than in the placebo group. In addition, we performed an exposure culture assay of inactivated influenza virus on peripheral blood mononuclear cells (PBMCs) isolated from the blood of participants in the HC and placebo groups. Gene-expression analysis in PBMCs after culture completion showed that IFNα and TLR7 expression levels were significantly higher in the HC group than in the placebo group. In addition, the expression levels of CD304 tended to be higher in the HC group than in the placebo group. On the other hand, the HC group showed a significantly higher increase in the intestinal butyrate concentration than the placebo group. HC intake also significantly suppressed levels of IL-6 and TNFα produced by PBMCs after exposure to inactivated influenza virus. Collectively, these results suggest that HC activated plasmacytoid dendritic cells expressing TLR7 and CD304 and strongly induced IFNα production, subsequently activating NK cells and increasing sIgA levels, and induced anti-inflammatory effects via increased intestinal butyrate levels. These changes may contribute to the acquisition of host resistance to viral infection and URTI prevention.

Antibody-dependent enhancement and neutralization against CVB4 investigated in vitro and in silico through an agent-based model.

The infection with coxsackievirus B4 (CVB4) can be enhanced in vitro by antibodies directed against the viral capsid protein VP4. In peripheral blood mononuclear cells, antibody-dependent enhancement (ADE) of CVB4 infection leads to the production of interferon alpha (IFN-α). To investigate ADE of CVB4-induced production of IFN-α, an agent-based model was constructed with enhancing and neutralizing antibodies. The model recapitulates viral neutralization and ADE in silico. The enhancing and neutralizing activities of serum samples were evaluated in vitro to confront the model predictions with experimental results. Increasing the incubation time of CVB4 with serum samples improves virus neutralization in silico as well as in vitro. It also results in ADE at lower antibody numbers in silico, which is confirmed in vitro with IFN-α production at lower serum concentrations. Furthermore, incubation of CVB4 with serum at a low temperature does not induce IFN-α production in vitro. Thus, taken together our results suggest that enhancing antibodies bind cryptic epitopes, more accessible with longer incubation time and at higher temperature due to changes in capsid conformation, consistent with previous results indicating that enhancing antibodies are anti-VP4 antibodies.

Interferon system in typical chronic and atypically occurring chronic active herpes virus infections

Objective: to study the characteristics of the functioning of the IFN system, the presence of autoantibodies to INFα in patients suffering from atypical chronic active herpesvirus infections (ACA-HVI) in comparison with patients with a typical course of chronic herpesvirus infections (CHVI).Materials and methods: under our supervision were 485 patients of both sexes aged 23 to 70 years, suffering from chronic herpes virus infections, of which 335 patients suffered from AHA-HVI and 150 people suffered from CHVI. The comparison group was 250 conditionally healthy individuals (CG). The complex of the study included methods for detecting herpesviruses: serodiagnostics, PCR-RT. The IFN system (spontaneous and induced production, serum concentration) was tested by ELISA. The study was approved by the ethics board and informed consent was obtained from all patients.Results: the incidence of various mono-mixed herpesvirus infections in patients with ACA-HVI (mono – 26,6% and mixed – 73.4%) and CHVI (mono – 23.1% and mixed – 76.9%) was determined, with EBV dominance in patients of both groups. Serum IFNα deficiency was detected in 100% of cases in both groups, and IFNα in 67% in ACA-HVI and 57% in CHVI. At the same time, significant differences were found between the ACA-HVI and HGVI groups in the level of IFNα reduction: 10 and 5 times, respectively, and for IFNγ – 2.0 and 2.6 times, respectively. The induced IFNα production decreased by 89.1% in ACA-HVI and 47.2% in CHVI. A decrease in induced IFNγ production is characteristic of 50% of patients in both groups. At the same time, the level of induced production of IFN α in patients with ACA-HVI was 9 times lower than in the control group and 4.75 times lower than in the group of patients with CHVI. And the level of induced IFNγ production was 2 times lower compared to CHVI and the control group.Conclusions: when assessing the state of the IFN system in patients with various chronic herpes virus infections, significant differences were revealed. Thus, the most pronounced manifestations of interferonopathy, consisting in a significant decrease in serum IFNα and IFNγ and defects in induced IFN production of both types, are observed statistically significantly more often in the group of patients with an atypical course of the disease than in the group of patients with a typical course of CHVI. The most pronounced disorders in the IFN system and the lack of recovery of IFNα and IFNγ levels in the interrelational period cause atypicity of the course and active viral replication in patients with ACA-HVI.

Singapore Grouper Iridovirus VP131 Drives Degradation of STING-TBK1 Pathway Proteins and Negatively Regulates Antiviral Innate Immunity.

Iridoviruses are large DNA viruses which cause great economic losses to the aquaculture industry and serious threats to ecological diversity worldwide. Singapore grouper iridovirus (SGIV), a novel member of the genus Ranavirus, causes high mortality in grouper aquaculture. Previous work on genome annotation demonstrated that SGIV contained numerous uncharacterized or hypothetical open reading frames (ORFs), whose functions remained largely unknown. Here, we reported that the protein encoded by SGIV ORF131R (VP131) was localized predominantly within the endoplasmic reticulum (ER). Ectopic expression of GFP-VP131 significantly enhanced SGIV replication, while VP131 knockdown decreased viral infection in vitro, suggesting that VP131 functioned as a proviral factor during SGIV infection. Overexpression of GFP-VP131 inhibited the interferon (IFN)-1 promoter activity and mRNA level of IFN-related genes induced by poly(I:C), Epinephelus coioides cyclic GMP/AMP synthase (EccGAS)/stimulator of IFN genes (EcSTING), TANK-binding kinase 1 (EcTBK1), or melanoma differentiation-associated gene 5 (EcMDA5), whereas such activation induced by mitochondrial antiviral signaling protein (EcMAVS) was not affected. Moreover, VP131 interacted with EcSTING and degraded EcSTING through both the autophagy-lysosome pathway and ubiquitin-proteasome pathway, and targeted for the K63-linked ubiquitination. Of note, we also found that EcSTING significantly accelerated the formation of GFP-VP131 aggregates in co-transfected cells. Finally, GFP-VP131 inhibited EcSTING- or EcTBK1-induced antiviral activity upon red-spotted grouper nervous necrosis virus (RGNNV) infection. Together, our results demonstrated that the SGIV VP131 negatively regulated the IFN response by inhibiting EcSTING-EcTBK1 signaling for viral evasion. IMPORTANCE STING has been identified as a critical factor participating in the innate immune response which recruits and phosphorylates TBK1 and IFN regulatory factor 3 (IRF3) to induce IFN production and defend against viral infection. However, viruses also distort the STING-TBK1 pathway to negatively regulate the IFN response and facilitate viral replication. Here, we reported that SGIV VP131 interacted with EcSTING within the ER and degraded EcSTING, leading to the suppression of IFN production and the promotion of SGIV infection. These results for the first time demonstrated that fish iridovirus evaded the host antiviral response via abrogating the STING-TBK1 signaling pathway.

Screening of interferon-stimulated genes against avian reovirus infection and mechanistic exploration of the antiviral activity of IFIT5.

Avian reovirus (ARV) infection can lead to severe immunosuppression, complications, and secondary diseases, causing immense economic losses to the poultry industry. In-depth study of the mechanism by which the innate immune system combats ARV infection, especially the antiviral effect mediated by interferon, is needed to prevent and contain ARV infection. In this study, ARV strain S1133 was used to artificially infect 7-day-old specific pathogen–free chickens. The results indicated that ARV rapidly proliferated in the immune organs, including the spleen, bursa of Fabricius, and thymus. The viral load peaked early in the infection and led to varying degrees of pathological damage to tissues and organs. Real-time quantitative PCR revealed that the mRNA levels of interferon and multiple interferon-stimulated genes (ISGs) in the spleen, bursa of Fabricius, and thymus were upregulated to varying degrees in the early stage of infection. Among the ISGs, IFIT5, and Mx were the most upregulated in various tissues and organs, suggesting that they are important ISGs for host resistance to ARV infection. Further investigation of the role of IFIT5 in ARV infection showed that overexpression of the IFIT5 gene inhibited ARV replication, whereas inhibition of the endogenously expressed IFIT5 gene by siRNA promoted ARV replication. IFIT5 may be a positive feedback regulator of the innate immune signaling pathways during ARV infection and may induce IFN-α production by promoting the expression of MAD5 and MAVS to exert its antiviral effect. The results of this study help explain the innate immune regulatory mechanism of ARV infection and reveal the important role of IFIT5 in inhibiting ARV replication, which has important theoretical significance and practical application value for the prevention and control of ARV infection.

Lactoferrin and its digestive peptides induce interferon-α production and activate plasmacytoid dendritic cells ex vivo

Plasmacytoid dendritic cells (pDCs) recognise viral single-stranded RNA (ssRNA) or CpG DNA via Toll-like receptor (TLR)-7 and TLR9, and produce interferon (IFN)-α. Activated pDCs upregulate human leukocyte antigen (HLA)-DR and CD86 expression levels. Ingestion of bovine lactoferrin (LF) activates pDCs, but little is known about its effects. In this study, the effects of LF and its pepsin hydrolysate (LFH) on the production of IFN-α from peripheral blood mononuclear cells (PBMCs) and pDCs were examined. PBMCs were prepared from peripheral blood of healthy adults and incubated with LF, LFH, or lactoferricin (LFcin) in the absence or presence of ssRNA derived from human immunodeficiency virus. The concentration of IFN-α in the supernatant and the expression levels of IFN-α, HLA-DR, and CD86 in pDCs were quantified by enzyme-linked immunosorbent assay and flow cytometry. In the absence of ssRNA, the concentration of IFN-α was negligible and LF had no effect on it. In the presence of ssRNA, IFN-α was detected at a certain level, and LF and LFH significantly increased its concentration. The increase caused by LFH and LFcin were comparable. In addition, LF significantly upregulated the expression levels of IFN-α, HLA-DR, and CD86 in pDCs. LF and its digestive peptides induced IFN-α production and activated pDCs in the presence of ssRNA, suggesting that LF modulates the immune system by promoting pDC activation upon viral recognition.

XIST is a source of TLR7 ligands underlying the sex bias in Systemic Lupus Erythematosus

Abstract Systemic Lupus Erythematosus (SLE) is among the most sex-biased autoimmune diseases identified to date, affecting 9-times more women than men. Recognition of self-RNA by Toll-like receptor 7 (TLR7) is implicated as a central pathogenic process leading to the aberrant production of type-I interferon (IFN) in SLE, but the specific RNA molecules contributing to this process have not been defined. Given the role of self-RNA and biological sex in SLE pathogenesis, we investigated which sex-biased self-RNAs are potentially responsible for aberrant TLR7 activation in SLE. We used recent revelations about TLR7 sequence specificity and publicly available RNA sequencing data to identify sex-biased sources of self-RNA containing TLR7 ligands. We found X-inactive specific transcript (XIST) to be a particularly rich source of TLR7 ligands that is specifically expressed in women. We then investigated the capacity of XIST to act as a TLR7 ligand in vitro and found that multiple fragments of XIST induce IFNα production in a TLR7-dependent manner more than control RNA of equal length. Furthermore, we found that RNA isolated from XIST-knockout cells have significantly reduced capacity to stimulate TLR7 compared to RNA from wild-type cells. Finally, we used flow cytometry and publicly available RNA sequencing data to investigate the connection between XIST and SLE disease variables. We found that higher XIST expression correlated with SLE disease status, higher SLE disease activity index (SLEDAI) scores, and the interferon signature. Our data suggest that XIST is a source of TLR7 ligands that may underlie the sex bias in SLE. Supported by grants from National Institute of Dental and Craniofacial Research (NIDCR), NIH (R21 DE028391-02), the Jerome L. Greene Foundation and Scleroderma Research Foundation.

Baculovirus Vectors Induce the Production of Interferons in Swine: Their Potential in the Development of Antiviral Strategies.

The huge variety of viruses affecting swine represents a global threat. Since vaccines against highly contagious viruses last several days to induce protective immune responses, antiviral strategies for rapid control of outbreak situations are needed. The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV), an insect virus, has been demonstrated to be an effective vaccine vector for mammals. Besides the ability to display or transduce heterologous antigens, it also induces strong innate immune responses and provides IFN-mediated protection against lethal challenges with viruses like foot-and-mouth disease virus (FMDV) in mice. Thus, the aim of this study was to evaluate the ability of AcMNPV to induce IFN production and elicit antiviral activity in porcine peripheral blood mononuclear cells (PBMCs). Our results demonstrated that AcMNPV induced an IFN-α-mediated antiviral activity in PBMCs in vitro. Moreover, the inoculation of AcMNPV in piglets led to the production of type I and II IFNs in sera from inoculated animals and antiviral activities against vesicular stomatitis virus (VSV) and FMDV measured by in vitro assays. Finally, it was demonstrated that the pseudotyping of AcMNPV with VSV-G protein, but not the enrichment of the AcMNPV genome with specific immunostimulatory CpG motifs for the porcine TLR9, improved the ability to induce IFN-α production in PBMCs in vitro. Together, these results suggest that AcMNPV is a promising tool for the induction of IFNs in antiviral strategies, with the potential to be biotechnologically improved.

Feeding with poly(I:C) induced long-term immune responses against bacterial infection in turbot (Scophthalmus maximus)

Poly(I:C) is a kind of chemosynthetic double-stranded RNA (dsRNA) analogue which could act as TLR3 agonist and induce IFN production. It is widely applied in anti-virus treatment and immunoregulation, as well as vaccine adjuvant in farmed animals. However, whether poly(I:C) could activate innate immune response to defense against bacterial infection remains unclear. In this study, we established a feeding trial model with different dose of poly(I:C) in turbot larvae, then challenged with Edwardsiella piscicida after 3–7 weeks resting period. The results show that feeding turbot with poly(I:C) exhibited a stronger inflammatory response and antioxidant stress ability, and significantly elevated the survival rate within the decreased bacterial loads. Importantly, the bacterial infection-induced white feces in hindgut of turbot were significantly alleviated after poly(I:C) feeding, and this administration induced protection could last for about 7 weeks. Taken together, these findings indicate that feeding turbot with poly(I:C) could enhance a long-term intestinal mucosal immunity in response to bacterial infection, suggesting that poly(I:C) might be a promising immunostimulant in aquaculture.

NETs decorated with bioactive IL-33 infiltrate inflamed tissues and induce IFN-α production in patients with SLE

IL-33, a nuclear alarmin released during cell death, exerts context-specific effects on adaptive and innate immune cells, eliciting potent inflammatory responses. We screened blood, skin, and kidney tissues from patients with systemic lupus erythematosus (SLE), a systemic autoimmune disease driven by unabated type I IFN production, and found increased amounts of extracellular IL-33 complexed with neutrophil extracellular traps (NETs), correlating with severe, active disease. Using a combination of molecular, imaging, and proteomic approaches, we show that SLE neutrophils, activated by disease immunocomplexes, release IL-33–decorated NETs that stimulate robust IFN-α synthesis by plasmacytoid DCs in a manner dependent on the IL-33 receptor ST2L. IL33-silenced neutrophil-like cells cultured under lupus-inducing conditions generated NETs with diminished interferogenic effect. Importantly, NETs derived from patients with SLE are enriched in mature bioactive isoforms of IL-33 processed by the neutrophil proteases elastase and cathepsin G. Pharmacological inhibition of these proteases neutralized IL-33–dependent IFN-α production elicited by NETs. We believe these data demonstrate a novel role for cleaved IL-33 alarmin decorating NETs in human SLE, linking neutrophil activation, type I IFN production, and end-organ inflammation, with skin pathology mirroring that observed in the kidneys.

Direct and indirect immune effects of CMP-001, a virus-like particle containing a TLR9 agonist

BackgroundCMP-001, also known as vidutolimod, is a virus-like particle containing a TLR9 agonist that is showing promise in early clinical trials. Our group previously demonstrated that the immunostimulatory effects of...

Zebrafish NIK Mediates IFN Induction by Regulating Activation of IRF3 and NF-κB.

Type I IFN mediates the innate immune system to provide defense against viral infections. NF-κB-inducing kinase (NIK) potentiates the basal activation of endogenous STING, which facilitates the recruitment of TBK1 with the ectopically expressed IRF3 to induce IFN production. Moreover, NIK phosphorylates IKKα and confers its ability to phosphorylate p100 (also known as NF-κB2) in mammals. Our study demonstrated that NIK plays a critical role in IFN production in teleost fish. It was found that NIK interacts with IKKα in the cytoplasm and that IKKα phosphorylates the NIK at the residue Thr432, which is different from the mammals. Overexpression of NIK caused the activation of IRF3 and NF-κB, which in turn led to the production of IFN and IFN-stimulated genes (ISGs). Furthermore, the ectopic expression of NIK was observed to be associated with a reduced replication of the fish virus, whereas silencing of endogenous NIK had an opposite effect in vitro. Furthermore, NIK knockdown significantly reduced the expression of IFN and key ISGs in zebrafish larvae after spring viremia of carp virus infection. Additionally, the replication of spring viremia of carp virus was enhanced in NIK knockdown zebrafish larvae, leading to a lower survival rate. In summary, our findings revealed a previously undescribed function of NIK in activating IFN and ISGs as a host antiviral response. These findings may facilitate the establishment of antiviral therapy to combat fish viruses.

Staphylococcus aureus Colonization Is Increased on Lupus Skin Lesions and Is Promoted by IFN-Mediated Barrier Disruption

Cutaneous inflammation is recurrent in systemic lupus erythematosus (SLE), yet mechanisms that drive cutaneous inflammation in SLE are not well defined. Type I IFNs are elevated in nonlesional SLE skin and promote inflammatory responses. Staphylococcus aureus, known to induce IFN production, could play a role in cutaneous inflammation in SLE. We show here that active cutaneous lupus erythematosus lesions are highly colonized (∼50%) by S.aureus. To define the impact of IFNs on S.aureus colonization, we examined the effects of type I and type II IFNs on S.aureus adherence and invasion. An increase in adherent S.aureus was observed after exposure to both IFN-α and -γ, whereas IFN-γ appeared to inhibit invasion of S.aureus. Cutaneous lupus erythematosus lesional skin microarray data and RNA sequencing data from SLE keratinocytes identified repression of barrier gene expression, such as filaggrin and loricrin, and SLE keratinocytes exhibited increased S.aureus-binding integrins. These SLE-associated changes could be replicated by IFN treatment of keratinocytes. Further, SLE keratinocytes exhibited increased binding to S.aureus. Together, these data suggest that chronic exposure to IFNs induces barrier disruption that allows for higher S.aureus colonization in SLE skin.

IRF7 Is Involved in Both STING and MAVS Mediating IFN-β Signaling in IRF3-Lacking Chickens

IFN regulatory factor (IRF) 3 has been identified as the most critical regulator of both RNA and DNA virus-induced IFN production in mammals. However, ambiguity exists in research on chicken IRFs; in particular IRF3 seems to be missing in chickens, making IFN regulation in chickens unclear. In this study, we comprehensively investigated the potential IFN-related IRFs in chickens and showed that IRF7 is the most critical IFN-β regulator in chickens. With a chicken IRF7 (chIRF7) knockout DF-1 cell line, we conducted a series of experiments to demonstrate that chIRF7 is involved in both chicken STING (chSTING)- and chicken MAVS (chMAVS)-mediated IFN-β regulation in response to DNA and RNA viral infections, respectively. We further examined the mechanisms of chIRF7 activation by chSTING. We found that chicken TBK1 (chTBK1) is indispensable for chIRF7 activation by chSTING as well as that chSTING interacts with both chIRF7 and chTBK1 to function as a scaffold in chIRF7 activation by chTBK1. More interestingly, we discovered that chSTING mediates the activation of chIRF7 through a conserved SLQxSyS motif. In short, we confirmed that although IRF3 is missing in chickens, they employ IRF7 to reconstitute corresponding IFN signaling to respond to both DNA and RNA viral infections. Additionally, we uncovered a mechanism of chIRF7 activation by chSTING. The results will enrich and deepen our understanding of the regulatory mechanisms of the chicken IFN system.

Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8)/CLM-6 is an FcR\u03b3-coupled receptor selectively expressed in mouse tissue plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) produce large amounts of type-I interferon (IFN) in response to viral infection or self nucleic acids. Leukocyte mono-immunoglobulin-like receptor 8 (LMIR8), also called CMRF-35-like molecule-6 (CLM-6), is a putative activating receptor among mouse LMIR/CLM/CD300 members; however, the expression and function of LMIR8 remain unclear. Here, we characterize mouse LMIR8 as a pDC receptor. Analysis of Flag-tagged LMIR8-transduced bone marrow (BM)-derived mast cells demonstrated that LMIR8 can transmit an activating signal by interacting with immunoreceptor tyrosine-based activating motif (ITAM)-containing FcRγ. Flow cytometric analysis using a specific antibody for LMIR8 showed that LMIR8 expression was restricted to mouse pDCs residing in BM, spleen, or lymph node. FcRγ deficiency dampened surface expression of LMIR8 in mouse pDCs. Notably, LMIR8 was detected only in pDCs, irrespective of TLR9 stimulation, suggesting that LMIR8 is a suitable marker for pDCs in mouse tissues; LMIR8 is weakly expressed in Flt3 ligand-induced BM-derived pDCs (BMpDCs). Crosslinking of transduced LMIR8 in BMpDCs with anti-LMIR8 antibody did not induce IFN-α production, but rather suppressed TLR9-mediated production of IFN-α. Taken together, these observations indicate that LMIR8 is an FcRγ-coupled receptor selectively expressed in mouse tissue pDCs, which might suppress pDC activation through the recognition of its ligands.

Tripartite motif-containing proteins precisely and positively affect host antiviral immune response.

The tripartite motif-containing proteins (TRIMs) comprise a large family of proteins with over 70 members in humans. Recent studies have shown that TRIMs play unexpected roles in the antiviral immune responses to infections by HIV, MLV, EMCV, AIV and other viruses. There are two mechanisms used by TRIMs in the inhibition of virus infections: (1) TRIMs target the produced viruses for ubiquitination, which induces proteasome-dependent degradation, or they interact with host proteins to inhibit viral infection in various periods of the viral life cycle. (2) TRIMs activate innate immune signalling pathways, such as RLR and TLR, which induce IFN production. In this study, we will review recent studies regarding the means by which TRIMs function as inhibitors in viral infection through the mechanisms described above.

Human innate responses and adjuvant activity of TLR ligands in vivo in mice reconstituted with a human immune system.

TLR ligands (TLR-Ls) represent a class of novel vaccine adjuvants. However, their immunologic effects in humans remain poorly defined in vivo. Using a humanized mouse model with a functional human immune system, we investigated how different TLR-Ls stimulated human innate immune response in vivo and their applications as vaccine adjuvants for enhancing human cellular immune response. We found that splenocytes from humanized mice showed identical responses to various TLR-Ls as human PBMCs in vitro. To our surprise, various TLR-Ls stimulated human cytokines and chemokines differently in vivo compared to that in vitro. For example, CpG-A was most efficient to induce IFN-α production in vitro. In contrast, CpG-B, R848 and Poly I:C stimulated much more IFN-α than CpG-A in vivo. Importantly, the human innate immune response to specific TLR-Ls in humanized mice was different from that reported in C57BL/6 mice, but similar to that reported in nonhuman primates. Furthermore, we found that different TLR-Ls distinctively activated and mobilized human plasmacytoid dendritic cells (pDCs), myeloid DCs (mDCs) and monocytes in different organs. Finally, we showed that, as adjuvants, CpG-B, R848 and Poly I:C can all enhance antigen specific CD4+ T cell response, while only R848 and Poly I:C induced CD8+ cytotoxic T cells response to a CD40-targeting HIV vaccine in humanized mice, correlated with their ability to activate human mDCs but not pDCs. We conclude that humanized mice serve as a highly relevant model to evaluate and rank the human immunologic effects of novel adjuvants in vivo prior to testing in humans.

Lethal Experimental Tick-Borne Encephalitis Infection: Influence of Two Strains with Similar Virulence on the Immune Response.

Tick-borne encephalitis virus (TBEV) is a tick-transmitted arbovirus that causes serious diseases in humans in Europe and Northern Asia. About 6000–10,000 cases are registered annually, and one-third of them lead to sequela with different degrees of severity. Two TBEV strains (Absettarov and EK-328) similar in virulence rate in laboratory mice were used to study pathogenesis and immune response upon lethal infection in mice. The strains differed in the dynamics of appearance of virus, IFNs and other cytokines in blood of mice, and ability to induce a cytokine storm in the terminal stages of disease and a non-sterile immunity. Moreover, the TBEV strains differed in characteristics of their interactions with DCs: level of reproduction in these cells, virus dose triggering IFN-α production, and impact on DCs' maturation. Infection of DCs with Absettarov strain led to IFN-α induction only at high multiplicity of infection (MOI), and an increased amount of the mature DCs with high adhesion activity and low-level of MHCII positive cells. While reproduction of the EK-328 strain in DCs was less efficient, a low dose of the virus induced IFN-α production and stimulated maturation of DCs with relatively low adhesive capacity, but with the high percentage of cells expressing MHCII molecules. Thus, the studied strains differed significantly in the impact on DCs' maturation and antigen presentation to CD4+ lymphocytes. Injection of low (103 PFU) and high (106 PFU) doses of both TBEV strains caused a lethal infection in mice. At the same time, the dose of the virus in the inoculum, regardless of the strain properties, affected the following virulence characteristics: the time of virus appearance in brain (day 4–5 vs. day 1 p.i.), time of IFN-α appearance in blood (10 h vs. 5 h p.i.), concentration of IFN-α in blood, and induction of IFN-α during infection of DCs. Therefore, virulent TBEV strains during lethal infection can interact differently with the host immune system, and the infectious dose has an impact on both: virus spread in the infected organism and immune response activation.

C/EBPβ Is a Critical Regulator of CML Stem Cell Differentiation and Exhaustion Induced By Interferon-α

Even in the era of tyrosine kinase inhibitors, eradication of chronic myeloid leukemia (CML) stem cells still remains to be a prerequisite for the complete cure of the disease. Interferon-α (IFNα), which has been used long for the treatment for chronic phase (CP)-CML, are now being re-evaluated since recent clinical trials showed that IFNα significantly improved the clinical outcome of patients treated with imatinib. IFNα promotes progression of cell cycle and differentiation of normal hematopoietic stem/progenitor cells (HSPCs) (Essers MA et al, Nature 2009; Sato T et al, Nat Med2009). However, its effect and mechanism of action on CML stem cells have not been investigated yet.CCAAT/enhancer binding protein β (C/EBPβ) is a transcription factor playing an essential role for emergency granulopoiesis. Under stressed conditions including infections and cytokine stimulations, prompt and sufficient supply of granulocytes is evoked by C/EBPβ, which promotes both proliferation and myeloid differentiation of HSPCs (Hirai H et al, Nat Immunol 2006; Satake S et al, J Immunol 2012). In addition, we have previously shown that (1) C/EBPβ is upregulated in HSPCs obtained from patients with CP-CML, (2) STAT5 is responsible for the upregulation, and (3) C/EBPβ promotes myeloid differentiation of CML stem cells (Hayashi Y et al, Leukemia2013). IFNα is one of the inflammatory cytokines, thus we hypothesized that C/EBPβ is involved in the effect of IFNα on CML stem cells, through promoting their differentiation and exhaustion. In this study, we investigated the molecular interplay between IFNα and C/EBPβ in CML stem cells.First, we searched for regulatory elements of Cebpb, which is responsible for C/EBPβ upregulation induced by BCR-ABL-STAT5 signaling. By ChIP-seq analysis of a HSC line, EML cells, we identified novel BCR-ABL-responsive STAT5 binding sites in 3' distal region of Cebpb. Interestingly, two STAT5 consensus motifs (TTCNNNGAA) are tandemly located in this region, which is highly conserved among various species including human. Enrichment of H3K27Ac histone mark within this region in BCR-ABL-expressing EML cells was more evident than in empty vector-transduced cells. These results suggest that this region is a critical enhancer required for BCR-ABL-dependent expression of C/EBPβ.Next, we explored whether IFNα induces C/EBPβ expression in EML cells and found that IFNα rapidly phosphorylated STAT5 as well as STAT1 and STAT3, and increased C/EBPβ expression irrespective of the presence of BCR-ABL. Notably, IFNα also recruited STAT5 to the 3’ distal enhancer region of Cebpb mentioned above, suggesting that this enhancer might also regulate cytokine-responsive C/EBPβ expression.In order to clarify involvement of C/EBPβ in the action of IFNα, KSL cells from WT or C/EBPβ KO mouse bone marrow (BM) cells were retrovirally transduced with BCR-ABL, and subjected to serial replating assay. IFNα promoted differentiation of WT CML stem cells toward myeloid cells, and reduced their replating ability. In contrast, C/EBPβ-deficient CML stem cells retained their immature status and colony-forming ability even in the presence of IFNα.Finally, we evaluated the in vivo effect of IFNα on CML stem cells by serial BM transplantation experiments. The first recipients of BCR-ABL-transduced BM cells were administrated with PolyI:C, which induces IFNα production in vivo. The frequencies and numbers of WT CML stem cells in BM of the first recipients were significantly decreased by administration of PolyI:C, and this effect was severely abolished when C/EBPβ was absent in CML stem cells (Figure). Notably, PolyI:C treatment prolonged the survival of the secondary recipient mice only in the presence of C/EBPβ in CML stem cells, suggesting that IFNα promotes exhaustion of CML stem cells through C/EBPβ.Collectively, these data demonstrate that IFNα upregulates C/EBPβ at least in part through recruiting STAT5 to 3’ distal enhancer and that C/EBPβ is the critical regulator of differentiation and exhaustion of CML stem cells induced by IFNα. Our basic study presented here will shed light on the molecular mechanisms involved in the clinical efficacy of IFNα on CML. [Display omitted] DisclosuresHirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.