Lymphotoxin Alpha-Deficient Mice Clear Persistent Rotavirus Infection after Local Generation of Mucosal IgA

Rotavirus is a major cause of pediatric diarrheal illness worldwide. To explore the role of organized intestinal lymphoid tissues in infection by and immunity to rotavirus, lymphotoxin alpha-deficient (LTα^−/−) mice that lack Peyer''s patches and mesenteric lymph nodes were orally infected with murine rotavirus. Systemic rotavirus was cleared within 10 days in both LTα^−/− and wild-type mice, and both strains developed early and sustained serum antirotavirus antibody responses. However, unlike wild-type mice, which resolved the intestinal infection within 10 days, LTα^−/− mice shed fecal virus for approximately 50 days after inoculation. The resolution of fecal virus shedding occurred concurrently with induction of intestinal rotavirus-specific IgA in both mouse strains. Induction of intestinal rotavirus-specific IgA in LTα^−/− mice correlated with the (late) appearance of IgA-producing plasma cells in the small intestine. This, together with the absence of rotavirus-specific serum IgA, implies that secretory rotavirus-specific IgA was produced locally. These findings indicate that serum IgG responses are insufficient and imply that local intestinal IgA responses are important for the clearance of rotavirus from intestinal tissues. Furthermore, they show that while LTα-dependent lymphoid tissues are important for the generation of IgA-producing B cells in the intestine, they are not absolutely required in the setting of rotavirus infection. Moreover, the induction of local IgA-producing B cell responses can occur late after infection and in an LTα-independent manner.     

The role of CD40-CD154 interaction in antiviral T cell-independent IgG responses

Polyomavirus (PyV) infection elicits protective T cell-independent (TI) IgG responses in T cell-deficient mice. The question addressed in this report is whether CD40 signaling plays a role in this TI antiviral IgG response. Because CD40 ligand (CD40L) can be expressed on numerous cell types in addition to activated T cells, it is possible that cells other than T cells provide CD40L to signal through CD40 on B cells and hence positively influence the antiviral TI IgG responses. In this study we show, by blocking CD40-CD40L interactions in vivo with anti-CD40L Ab treatment in TCR betaxdelta-/- mice and by using SCID mice reconstituted with CD40-/- B cells, that the lack of CD40 signaling in B cells results in a 50% decrease in TI IgG secreted in response to PyV. SCID mice reconstituted with CD40L-/- B cells also responded to PyV infection with diminished IgG secretion compared with that of SCID mice reconstituted with wild-type B cells. This finding suggests that B cells may provide the CD40L for CD40 signaling in the absence of T cell help during acute virus infection. Our studies demonstrate that, although about half of the TI IgG responses to PyV are independent of CD40-CD40L interactions, these interactions occur in T cell-deficient mice and enhance antiviral TI Ab responses.     

IgG Suppresses Antibody Responses in Mice Lacking C1q,C3, Complement Receptors 1 and 2, or IgG Fc-Receptors

Antigen-specific IgG antibodies, passively administered to mice or humans together with large particulate antigens like erythrocytes, can completely suppress the antibody response against the antigen. This is used clinically in Rhesus prophylaxis, where administration of IgG anti-RhD prevents RhD-negative women from becoming immunized against RhD-positive fetal erythrocytes aquired transplacentally. The mechanisms by which IgG suppresses antibody responses are poorly understood. We have here addressed whether complement or Fc-receptors for IgG (FcγRs) are required for IgG-mediated suppression. IgG, specific for sheep red blood cells (SRBC), was administered to mice together with SRBC and the antibody responses analyzed. IgG was able to suppress early IgM- as well as longterm IgG-responses in wildtype mice equally well as in mice lacking FcγRIIB (FcγRIIB knockout mice) or FcγRI, III, and IV (FcRγ knockout mice). Moreover, IgG was able to suppress early IgM responses equally well in mice lacking C1q (C1qA knockout mice), C3 (C3 knockout mice), or complement receptors 1 and 2 (Cr2 knockout mice) as in wildtype mice. Owing to the previously described severely impaired IgG responses in the complement deficient mice, it was difficult to assess whether passively administered IgG further decreased their IgG response. In conclusion, Fc-receptor binding or complement-activation by IgG does not seem to be required for its ability to suppress antibody responses to xenogeneic erythrocytes.     

Trivalent Combination Vaccine Induces Broad Heterologous Immune Responses to Norovirus and Rotavirus in Mice

Rotavirus (RV) and norovirus (NoV) are the two major causes of viral gastroenteritis (GE) in children worldwide. We have developed an injectable vaccine design to prevent infection or GE induced with these enteric viruses. The trivalent combination vaccine consists of NoV capsid (VP1) derived virus-like particles (VLPs) of GI-3 and GII-4 representing the two major NoV genogroups and tubular RV recombinant VP6 (rVP6), the most conserved and abundant RV protein. Each component was produced in insect cells by a recombinant baculovirus expression system and combined in vitro. The vaccine components were administered intramuscularly to BALB/c mice either separately or in the trivalent combination. High levels of NoV and RV type specific serum IgGs with high avidity (>50%) as well as intestinal IgGs were detected in the immunized mice. Cross-reactive IgG antibodies were also elicited against heterologous NoV VLPs not used for immunization (GII-4 NO, GII-12 and GI-1 VLPs) and to different RVs from cell cultures. NoV-specific serum antibodies blocked binding of homologous and heterologous VLPs to the putative receptors, histo-blood group antigens, suggesting broad NoV neutralizing activity of the sera. Mucosal antibodies of mice immunized with the trivalent combination vaccine inhibited RV infection in vitro. In addition, cross-reactive T cell immune responses to NoV and RV-specific antigens were detected. All the responses were sustained for up to six months. No mutual inhibition of the components in the trivalent vaccine combination was observed. In conclusion, the NoV GI and GII VLPs combination induced broader cross-reactive and potentially neutralizing immune responses than either of the VLPs alone. Therefore, trivalent vaccine might induce protective immune responses to the vast majority of circulating NoV and RV genotypes.     

Expression of Suppressor of Cytokine Signaling 1 (SOCS1) Impairs Viral Clearance and Exacerbates Lung Injury during Influenza Infection

Suppressor of cytokine signaling (SOCS) proteins are inducible feedback inhibitors of cytokine signaling. SOCS1^−/− mice die within three weeks postnatally due to IFN-γ-induced hyperinflammation. Since it is well established that IFN-γ is dispensable for protection against influenza infection, we generated SOCS1^−/−IFN-γ^−/− mice to determine whether SOCS1 regulates antiviral immunity in vivo. Here we show that SOCS1^−/−IFN-γ^−/− mice exhibited significantly enhanced resistance to influenza infection, as evidenced by improved viral clearance, attenuated acute lung damage, and consequently increased survival rates compared to either IFN-γ^−/− or WT animals. Enhanced viral clearance in SOCS1^−/−IFN-γ^−/− mice coincided with a rapid onset of adaptive immune responses during acute infection, while their reduced lung injury was associated with decreased inflammatory cell infiltration at the resolution phase of infection. We further determined the contribution of SOCS1-deficient T cells to antiviral immunity. Anti-CD4 antibody treatment of SOCS1^−/−IFN-γ^−/− mice had no significant effect on their enhanced resistance to influenza infection, while CD8⁺ splenocytes from SOCS1^−/−IFN-γ^−/− mice were sufficient to rescue RAG1^−/− animals from an otherwise lethal infection. Surprisingly, despite their markedly reduced viral burdens, RAG1^−/− mice reconstituted with SOCS1^−/−IFN-γ^−/− adaptive immune cells failed to ameliorate influenza-induced lung injury. In conclusion, in the absence of IFN-γ, the cytoplasmic protein SOCS1 not only inhibits adaptive antiviral immune responses but also exacerbates inflammatory lung damage. Importantly, these detrimental effects of SOCS1 are conveyed through discrete cell populations. Specifically, while SOCS1 expression in adaptive immune cells is sufficient to inhibit antiviral immunity, SOCS1 in innate/stromal cells is responsible for aggravated lung injury.     

Dectin-2-Dependent NKT Cell Activation and Serotype-Specific Antibody Production in Mice Immunized with Pneumococcal Polysaccharide Vaccine

Although thymus-independent type 2 antigens generally do not undergo Ig class switching from IgM to IgG, pneumococcal polysaccharide vaccine (PPV) induces the production of serotype-specific IgG. How this happens remains unclear, however. In the present study, PPV immunization induced production of IgG as well as IgM specific for a serotype 3-pneumococcal polysaccharide in the sera of wild-type (WT) mice, but this phenomenon was significantly reduced in Dectin-2 knockout (KO) mice. Immunization with PPV caused IL-12p40 production in WT mice, but this response was significantly reduced in Dectin-2KO mice. Likewise, immunization with PPV activated natural killer T (NKT) cells in WT mice but not in Dectin-2KO mice. Furthermore, administration of α-galactosylceramide, recombinant (r)IL-12 or rIFN-γ improved the reduced IgG levels in Dectin-2KO mice, and treatment with neutralizing anti-IFN-γ mAb resulted in the reduction of IgG synthesis in PPV-immunized WT mice. Transfer of spleen cells from PPV-immunized WT mice conferred protection against pneumococcal infection on recipient mice, whereas this effect was cancelled when the transferred spleen cells were harvested from PPV-immunized Dectin-2KO mice. These results suggest that the detection of PPV antigens via Dectin-2 triggers IL-12 production, which induces IFN-γ synthesis by NKT cells and subsequently the production of serotype-specific IgG.     

Gamma Interferon Regulates Contraction of the Influenza Virus-Specific CD8 T Cell Response and Limits the Size of the Memory Population

The factors that regulate the contraction of the CD8 T cell response and the magnitude of the memory cell population against localized mucosal infections such as influenza are important for generation of efficient vaccines but are currently undefined. In this study, we used a mouse model of influenza to demonstrate that the absence of gamma interferon (IFN-γ) or IFN-γ receptor 1 (IFN-γR1) leads to aberrant contraction of antigen-specific CD8 T cell responses. The increased accumulation of the effector CD8 T cell population was independent of viral load. Reduced contraction was associated with an increased fraction of CD8 T cells expressing the interleukin-7 receptor (IL-7R) at the peak of the response, resulting in enhanced numbers of memory/memory precursor cells in IFN-γ^−/− and IFN-γR^−/− compared to wild-type (WT) mice. Blockade of IL-7 within the lungs of IFN-γ^−/− mice restored the contraction of influenza virus-specific CD8 T cells, indicating that IL-7R is important for survival and is not simply a consequence of the lack of IFN-γ signaling. Finally, enhanced CD8 T cell recall responses and accelerated viral clearance were observed in the IFN-γ^−/− and IFN-γR^−/− mice after rechallenge with a heterologous strain of influenza virus, confirming that higher frequencies of memory precursors are formed in the absence of IFN-γ signaling. In summary, we have identified IFN-γ as an important regulator of localized viral immunity that promotes the contraction of antigen-specific CD8 T cells and inhibits memory precursor formation, thereby limiting the size of the memory cell population after an influenza virus infection.     

Both TRIF and IPS-1 Adaptor Proteins Contribute to the Cerebral Innate Immune Response against Herpes Simplex Virus 1 Infection

Toll-like receptors (TLRs) and RNA helicases (RLHs) are important cell sensors involved in the immunological control of viral infections through production of type I interferon (IFN). The impact of a deficiency in the TRIF and IPS-1 adaptor proteins, respectively, implicated in TLR3 and RLH signaling pathways, was investigated during herpes simplex virus 1 (HSV-1) encephalitis. TRIF^−/−, IPS-1^−/−, and C57BL/6 wild-type (WT) mice were infected intranasally with 7.5 × 10⁵ PFU of HSV-1. Mice were monitored for neurological signs and survival over 20 days. Groups of mice were sacrificed on days 3, 5, 7, 9, and 11 postinfection for determination of brain viral replication by quantitative PCR (qPCR), plaque assay, and immunohistochemistry and for alpha/beta interferon (IFN-α/β) levels and phosphorylation of interferon regulatory factors 3 and 7 (IRF-3 and -7) in brain homogenates by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. TRIF^−/− and IPS-1^−/− mice had higher mortality rates than WT mice (P = 0.02 and P = 0.09, respectively). Viral antigens were more disseminated throughout the brain, correlating with a significant increase in brain viral load for TRIF^−/− (days 5 to 9) and IPS-1^−/− (days 7 and 9) mice compared to results for the WT. IFN-β production was reduced in brain homogenates of TRIF^−/− and IPS-1^−/− mice on day 5 compared to results for the WT, whereas IFN-α levels were increased on day 7 in TRIF^−/− mice. Phosphorylation levels of IRF-3 and IRF-7 were decreased in TRIF^−/− and IPS-1^−/− mice, respectively. These data suggest that both the TRIF and IPS-1 signaling pathways are important for the control of HSV replication in the brain and survival through IFN-β production.     

In Vivo Immune Evasion Mediated by the Herpes Simplex Virus Type 1 Immunoglobulin G Fc Receptor

Herpes simplex virus (HSV) glycoproteins gE and gI form an immunoglobulin G (IgG) Fc receptor (FcγR) that binds the Fc domain of human anti-HSV IgG and inhibits Fc-mediated immune functions in vitro. gE or gI deletion mutant viruses are avirulent, probably because gE and gI are also involved in cell-to-cell spread. In an effort to modify FcγR activity without affecting other gE functions, we constructed a mutant virus, NS-gE₃₃₉, that has four amino acids inserted into gE within the domain homologous to mammalian IgG FcγRs. NS-gE₃₃₉ expresses gE and gI, is FcγR⁻, and does not participate in antibody bipolar bridging since it does not block activities mediated by the Fc domain of anti-HSV IgG. In vivo studies were performed with mice because the HSV-1 FcγR does not bind murine IgG; therefore, the absence of an FcγR should not affect virulence in mice. NS-gE₃₃₉ causes disease at the skin inoculation site comparably to wild-type and rescued viruses, indicating that the FcγR⁻ mutant virus is pathogenic in animals. Mice were passively immunized with human anti-HSV IgG and then infected with mutant or wild-type virus. We postulated that the HSV-1 FcγR should protect wild-type virus from antibody attack. Human anti-HSV IgG greatly reduced viral titers and disease severity in NS-gE₃₃₉-infected animals while having little effect on wild-type or rescued virus. We conclude that the HSV-1 FcγR enables the virus to evade antibody attack in vivo, which likely explains why antibodies are relatively ineffective against HSV infection.     

Resistance of Interleukin-1β-Deficient Mice to Fatal Sindbis Virus Encephalitis

Interleukin-1β (IL-1β) concentrations are frequently elevated in central nervous system (CNS) viral infections, but the pathophysiologic significance of such elevations is not known. To examine the role of IL-1β in CNS viral pathogenesis, we compared the natural histories of IL-1β-deficient and wild-type 129 SV(ev) mice infected with a neurovirulent viral strain, neuroadapted Sindbis virus (NSV). We found that the incidence of severe paralysis and death was markedly decreased in NSV-infected IL-1β^−/− mice compared to NSV-infected wild-type mice (4 versus 88%, P < 0.001). Despite this marked difference in clinical outcome, no differences in numbers of apoptotic cells or presence of histopathologic lesions in the brains of moribund wild-type mice and those of clinically healthy IL-1β^−/− mice could be detected. These results suggest that IL-1β deficiency is protective against fatal Sindbis virus infection by a mechanism that does not involve resistance to CNS virus-induced apoptosis or histopathology.     

Ifit2 Deficiency Results in Uncontrolled Neurotropic Coronavirus Replication and Enhanced Encephalitis via Impaired Alpha/Beta Interferon Induction in Macrophages

Type I interferons (IFN-α/β) limit viral dissemination prior to the emergence of adaptive immune responses through the concerted action of interferon-stimulated genes (ISGs). Although IFN-α/β induction by coronaviruses is modest, it effectively limits viral spread within the central nervous system (CNS) and protects against mortality. The protective roles of specific ISGs against the mouse hepatitis virus (MHV) members of the coronaviruses are largely unknown. This study demonstrates a protective role of the ISG Ifit2 in encephalitis induced by the dual hepato- and neurotropic MHV-A59. Contrasting the mild encephalitis and 100% survival of MHV-A59-infected wild-type (wt) mice, nearly 60% of infected Ifit2^−/− mice exhibited severe encephalitis and succumbed between 6 and 8 days postinfection. Increased clinical disease in Ifit2^−/− mice coincided with higher viral loads and enhanced viral spread throughout the CNS parenchyma. Ifit2^−/− mice also expressed significantly reduced IFN-α/β and downstream ISG mRNAs Ifit1, Isg15, and Pkr, while expression of proinflammatory cytokines and chemokines was only modestly affected in the CNS. Impaired IFN-α/β induction in the absence of Ifit2 was confirmed by ex vivo mRNA analysis of microglia and macrophages, the prominent cell types producing IFN-α/β following MHV CNS infection. Furthermore, both IFN-α/β mRNA and protein production were significantly reduced in MHV-infected Ifit2^−/− relative to wt bone marrow-derived macrophages. Collectively, the data implicate Ifit2 as a positive regulator of IFN-α/β expression, rather than direct antiviral mediator, during MHV-induced encephalitis.     

Murine Coronavirus-Induced Subacute Fatal Peritonitis in C57BL/6 Mice Deficient in Gamma Interferon

Gamma interferon-deficient (IFN-γ−/−) mice with a C57BL/6 background were infected intraperitoneally with mouse hepatitis virus strain JHM (JHMV). In contrast to IFN-γ-+/− and IFN-γ+/+ mice, JHMV persisted in IFN-γ−/− mice and induced death during the subacute phase of the infection. Unexpectedly, infected IFN-γ−/− mice showed severe peritonitis accompanying the accumulation of a viscous fluid in the abdominal and thoracic cavities in the subacute phase. Destructive changes of hepatocytes were not observed. Administration of recombinant IFN-γ protracted the survival time of IFN-γ−/− mice after JHMV infection. These results demonstrate that IFN-γ plays a critical role in viral clearance in JHMV infection. They also show that a resultant persistent JHMV infection induces another form of disease in IFN-γ−/− mice, which bears a resemblance to feline infectious peritonitis in cats.     

RNase L Mediated Protection from Virus Induced Demyelination

IFN-α/β plays a critical role in limiting viral spread, restricting viral tropism and protecting mice from neurotropic coronavirus infection. However, the IFN-α/β dependent mechanisms underlying innate anti-viral functions within the CNS are poorly understood. The role of RNase L in viral encephalomyelitis was explored based on its functions in inhibiting translation, inducing apoptosis, and propagating the IFN-α/β pathway through RNA degradation intermediates. Infection of RNase L deficient (RL^−/−) mice with a sub-lethal, demyelinating mouse hepatitis virus variant revealed that the majority of mice succumbed to infection by day 12 p.i. However, RNase L deficiency did not affect overall control of infectious virus, or diminish IFN-α/β expression in the CNS. Furthermore, increased morbidity and mortality could not be attributed to altered proinflammatory signals or composition of cells infiltrating the CNS. The unique phenotype of infected RL^−/− mice was rather manifested in earlier onset and increased severity of demyelination and axonal damage in brain stem and spinal cord without evidence for enhanced neuronal infection. Increased tissue damage coincided with sustained brain stem infection, foci of microglia infection in grey matter, and increased apoptotic cells. These data demonstrate a novel protective role for RNase L in viral induced CNS encephalomyelitis, which is not reflected in overall viral control or propagation of IFN-α/β mediated signals. Protective function is rather associated with cell type specific and regional restriction of viral replication in grey matter and ameliorated neurodegeneration and demyelination.     

Reinvestigating the Role of IgM in Rabies Virus Postexposure Vaccination

B cells secreting IgG antibodies, but not IgM, are thought to be solely responsible for vaccine-induced protection against rabies virus (RABV) infections in postexposure settings. In this report, we reinvestigated the potential for IgM to mediate protection in a mouse model of RABV vaccination. Immunocompetent mice immunized with an experimental live replication-deficient RABV-based vaccine produced virus neutralizing antibodies (VNAs) within 3 days of vaccination. However, mice unable to produce soluble IgM (sIgM^−/−) did not produce VNAs until 7 days postimmunization. Furthermore, sIgM^−/− mice were not protected against RABV infection when challenged 3 days postimmunization, while all wild-type mice survived challenge. Consistent with the lack of protection against pathogenic RABV challenge, approximately 50- to 100-fold higher viral loads of challenge virus were detected in the muscle, spinal cord, and brain of immunized sIgM^−/− mice compared to control mice. In addition, IgG antibody titers in vaccinated wild-type and sIgM^−/− mice were similar at all time points postimmunization, suggesting that protection against RABV challenge is due to the direct effects of IgM and not the influence of IgM on the development of effective IgG antibody titers. In all, early vaccine-induced IgM can limit dissemination of pathogenic RABV to the central nervous system and mediate protection against pathogenic RABV challenge. Considering the importance for the rapid induction of VNAs to protect against RABV infections in postexposure prophylaxis settings, these findings may help guide the development of a single-dose human rabies vaccine.     

Surfactant Protein-A Modulates LPS-Induced TLR4 Localization and Signaling via β-Arrestin 2

The soluble C-type lectin surfactant protein (SP)-A mediates lung immune responses partially via its direct effects on alveolar macrophages (AM), the main resident leukocytes exposed to antigens. SP-A modulates the AM threshold of lipopolysaccharide (LPS) activity towards an anti-inflammatory phenotype both in vitro and in vivo through various mechanisms. LPS responses are tightly regulated via distinct pathways including subcellular TLR4 localization and thus ligand sensing. The cytosolic scaffold and signaling protein β-arrestin 2 acts as negative regulator of LPS-induced TLR4 activation. Here we show that SP-A neither increases TLR4 abundancy nor co-localizes with TLR4 in primary AM. SP-A significantly reduces the LPS-induced co-localization of TLR4 with the early endosome antigen (EEA) 1 by promoting the co-localization of TLR4 with the post-Golgi compartment marker Vti1b in freshly isolated AM from rats and wild-type (WT) mice, but not in β-arrestin 2^−/− AM. Compared to WT mice pulmonary LPS-induced TNF-α release in β-arrestin 2^−/− mice is accelerated and enhanced and exogenous SP-A fails to inhibit both lung LPS-induced TNF-α release and TLR4/EEA1 positioning. SP-A, but not LPS, enhances β-arrestin 2 protein expression in a time-dependent manner in primary rat AM. The constitutive expression of β-arrestin 2 in AM from SP-A^−/− mice is significantly reduced compared to SP-A^+/+ mice and is rescued by SP-A. Prolonged endosome retention of LPS-induced TLR4 in AM from SP-A^−/− mice is restored by exogenous SP-A, and is antagonized by β-arrestin 2 blocking peptides. LPS induces β-arrestin 2/TLR4 association in primary AM which is further enhanced by SP-A. The data demonstrate that SP-A modulates LPS-induced TLR4 trafficking and signaling in vitro and in vivo engaging β-arrestin 2.     

Immunogenicity and virus-like particle formation of rotavirus capsid proteins produced in transgenic plants

The human pathogen, group A rotavirus, is the most prevalent cause of acute infantile and pediatric gastroenteritis worldwide, especially in developing countries. There is an urgent demand for safer, more effective and cheaper vaccines against rotavirus infection. Plant-derived antigens may provide an exclusive way to produce economical subunit vaccines. Virus-like particles, constituting viral capsid proteins without viral nucleic acids, are considered a far safer candidate compared with live attenuated viral vaccines. In this study, the rotavirus capsid proteins VP2, VP6 and VP7 were co-expressed in transgenic tobacco plants, and their expression levels, formation of rotavirus-like particles (RV VLPs) and immunogenicity were extensively studied. Quantitative real-time RT-PCR and Western blot analysis revealed that the expression level of vp6 was the highest while vp7 was expressed at the lowest levels. The RV VLPs were purified from transgenic tobacco plants and analyzed by electron microscopy and Western blot. Results indicated that the plant-derived VP2, VP6 and VP7 proteins self-assembled into 2/6 or 2/6/7 RV VLPs with a diameter of 60–80 nm. When orally delivered into mice with cholera toxin as an adjuvant, the total soluble protein extracted from transgenic tobacco plants induced rotavirus-specific antibodies comparable with those of attenuated rotavirus vaccines, while VP 2/6/7 induced higher serum IgG and fecal IgA titers compared with VP 2/6.     

Efficient, Repeated Adenovirus-Mediated Gene Transfer in Mice Lacking both Tumor Necrosis Factor Alpha and Lymphotoxin α

The efficiency of adenovirus-mediated gene transfer is now well established. However, the cellular and the humoral immune responses triggered by vector injection lead to the rapid elimination of the transduced cells and preclude any efficient readministration. The present investigation focuses on the role of tumor necrosis factor alpha (TNF-α), a proinflammatory cytokine, and the related cytokine lymphotoxin α (LTα), in mounting an immune reaction against recombinant adenovirus vectors. After gene transfer in the liver, mice genetically deficient for both cytokines (TNF-α/LTα^−/−), in comparison with normal mice, presented a weak acute-phase inflammatory reaction, a reduction in cellular infiltrates in the liver, and a severely impaired T-cell proliferative response to both Adenoviral and transgene product antigens. Moreover, we observed a strong reduction in the humoral response to the vector and the transgene product, with a drastic reduction of anti-adenovirus immunoglobulin A and G antibody isotypes. In addition, the reduction in antibody response observed in TNF-α/LTα^−/− and TNF-α/LTα^+/− mice versus TNF-α/LTα^+/+ mice links antibody levels to TNF-α/LTα gene dosage. Due to the absence of neutralizing antibodies, the TNF-α/LTα knockout mice successfully express a second gene transduced by a second vector injection. The discovery of the pivotal role played by TNF-α in controlling the antibody response against adenovirus will allow more efficient adenovirus-based strategies for gene therapy to be proposed.     

Herpes simplex virus type 2-induced mortality following genital infection is blocked by anti-tumor necrosis factor alpha antibody in CXCL10-deficient mice

The role of tumor necrosis factor alpha (TNF-α) was evaluated for CXCL10-deficient (CXCL10^−/−) mice which succumbed to genital herpes simplex virus type 2 (HSV-2) infection and possessed elevated levels of virus and TNF-α but not other cytokines in the central nervous system (CNS) and vaginal tissue within the first 7 days following virus exposure. Anti-TNF-α but not control antibody treatment offsets the elevated mortality rate of CXCL10^−/− mice, despite increased CNS viral titers. In addition, TNF-α neutralization suppressed recruitment of leukocyte subpopulations into the CNS, which is associated with reduced CCL2 and CXCL9 expression. Collectively, the results implicate TNF-α as the principal mediator of mortality in response to genital HSV-2 infection.     

Epithelial PBLD attenuates intestinal inflammatory response and improves intestinal barrier function by inhibiting NF-κB signaling

Intestinal barrier function defects and dysregulation of intestinal immune responses are two key contributory factors in the pathogenesis of ulcerative colitis (UC). Phenazine biosynthesis-like domain-containing protein (PBLD) was recently identified as a tumor suppressor in gastric cancer, hepatocellular carcinoma, and breast cancer; however, its role in UC remains unclear. Therefore, we analyzed colonic tissue samples from patients with UC and constructed specific intestinal epithelial PBLD-deficient (PBLD^IEC−/−) mice to investigate the role of this protein in UC pathogenesis. We found that epithelial PBLD was decreased in patients with UC and was correlated with levels of tight junction (TJ) and inflammatory proteins. PBLD^IEC−/− mice were more susceptible to dextran sulfate sodium (DSS)- and 2,4,6-trinitrobenzene sulfonic acid-induced colitis compared with wild-type (WT) mice. In DSS-induced colitis, PBLD^IEC−/− mice had impaired intestinal barrier function and greater immune cell infiltration in colonic tissue than WT mice. Furthermore, TJ proteins were markedly reduced in PBLD^IEC−/− mice compared with WT mice with colitis. Nuclear factor (NF)-κB activation was markedly elevated and resulted in higher expression levels of downstream effectors (C–C motif chemokine ligand 20, interleukin [IL]-1β, IL-6, and tumor necrosis factor [TNF]-α) in colonic epithelial cells isolated from PBLD^IEC−/− mice than WT mice with colitis. PBLD overexpression in intestinal epithelial cells (IECs) consistently inhibited TNF-α/interferon-γ-induced intestinal barrier disruption and TNF-α-induced inflammatory responses via the suppression of NF-κB. In addition, IKK inhibition (IKK-16) rescued excessive inflammatory responses induced by TNF-α in PBLD knockdown FHC cells. Co-immunoprecipitation assays showed that PBLD may interact with IKKα and IKKβ, thus inhibiting NF-κB signaling, decreasing inflammatory mediator production, attenuating colonic inflammation, and improving intestinal barrier function. Modulating PBLD expression may provide a novel approach for treatment in patients with UC.Subject terms: Ulcerative colitis, Chronic inflammation     

CD8α Dendritic Cells Drive Establishment of HSV-1 Latency

It is generally accepted that CD8 T cells play the key role to maintain HSV-1 latency in trigeminal ganglia of ocularly infected mice. Yet, comparably little is known about the role of innate immunity in establishment of viral latency. In the current study, we investigated whether CD8α DCs impact HSV-1 latency by examining latency in the trigeminal ganglia (TG) of wild-type (WT) C57BL/6 versus CD8α^−/− (lack functional CD8 T cells and CD8α⁺ DCs), CD8β^−/− (have functional CD8α⁺ T cells and CD8α⁺ DCs), and β2m^−/− (lack functional CD8 T cells but have CD8α⁺ DCs) mice as well as BXH2 (have functional CD8 T cells but lack CD8α⁺ DCs) versus WT C3H (have functional CD8α T cells and CD8α⁺ DCs) mice. We also determined whether the phenotype of CD8α^−/− and BXH2 mice could be restored to that of WT mice by adoptive transfer of WT CD8⁺ T cells or bone marrow (BM) derived CD8α⁺ DCs. Our results clearly demonstrate that CD8α DCs, rather than CD8 T cells, are responsible for enhanced viral latency and recurrences.