The roles of perforin- and Fas-dependent cytotoxicity in protection against cytopathic and noncytopathic viruses

In vitro, T cell-dependent cytotoxicity is mediated by two distinct mechanisms, one being perforin-, the other Fas-dependent. The contribution of both of these mechanisms to clearance of viral infections was investigated in mice for the noncytopathic lymphocytic choriomeningitis virus (LCMV) and the cytopathic vaccinia, vesicular stomatitis (VSV) and Semliki forest (SFV) viruses. Clearance of an acute LCMV infection was mediated by the perforin-dependent mechanism without measurable involvement of the Fas-dependent pathway. For the resolution of vaccinia virus infection and for resistance against VSV and SFV, however, neither of the two pathways was required. These data suggest that perforin-dependent cytotoxicity mediated by T cells is crucial for protection against noncytopathic viruses, whereas infections with cytopathic viruses are controlled by nonlytic T cell-dependent soluble mediators such as cytokines (IFN-γ against vaccinia virus) and neutralizing antibodies (against VSV and SFV).     

Engineering of a recombinant trivalent single-chain variable fragment antibody directed against rabies virus glycoprotein G with improved neutralizing potency

Human and equine rabies immunoglobulins are currently available for passive immunization against rabies. However, these are hampered by the limited supply and some drawbacks. Advances in antibody engineering have led to overcome issues of clinical applications and to improve the protective efficacy. In the present study, we report the generation of a trivalent single-chain Fv (scFv50AD1-Fd), that recognizes the rabies virus glycoprotein, genetically fused to the trimerization domain of the bacteriophage T4 fibritin, termed ‘foldon’ (Fd). scFv50AD1-Fd was expressed as soluble recombinant protein in bacterial periplasmic space and purified through affinity chromatography. The molecular integrity and stability were analyzed by polyacrylamide gradient-gel electrophoresis, size-exclusion chromatography and incubation in human sera. The antigen-binding properties of the trimeric scFv were analyzed by direct and competitive-ELISA. Its apparent affinity constant was estimated at 1.4 ± 0.25 × 10⁹ M⁻¹ and was 75-fold higher than its monovalent scFv (1.9 ± 0.68 × 10⁷ M⁻¹). The scFv50AD1-Fd neutralized rabies virus in a standard in vitro and in vivo neutralization assay. We showed a high neutralization activity up to 75-fold compared with monovalent format and the WHO standard serum. The gain in avidity resulting from multivalency along with an improved biological activity makes the trivalent scFv50AD1-Fd construct an important reagent for rabies protection. The antibody engineering approach presented here may serve as a strategy for designing a new generation of anti-rabies for passive immunotherapy.     

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3′ untranslated region (3′UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) ^−/− present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del^−/− mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del^−/− mice. ARE-Del^+/− mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del^+/− mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220⁺ cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del^−/− mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.     

A recombinant single chain antibody neutralizes coronavirus infectivity but only slightly delays lethal infection of mice

The variable region genes of a murine anti-coronavirus monoclonal antibody (mAb) were joined by assembly polymerase chain reaction and expressed in Escherichia coli in a single chain variable fragment (scFv) configuration. After induction of expression, the expected 32-kDa protein was identified by Western immunoblotting with specific rabbit anti-idiotype antibodies. The scFv fragments were purified from soluble cytoplasmic preparations by affinity chromatography on nickel agarose, which was possible with an N-terminal but not with a C-terminal histidine tag. Purified scFv fragments retained the antigen-binding properties of the parental antibody, could inhibit its binding to viral antigens with apparently higher efficiency than monovalent antigen-binding (Fab) fragments, but neutralized viral infectivity with lower efficiency (about sevenfold at a molar level). To evaluate the usefulness of these smaller and less immunogenic molecules in the treatment of viral diseases, mice were treated with purified recombinant scFv fragments and challenged with a lethal viral dose. A small delay in mortality was observed for the scFv-treated animals. Therefore, even though the scFv could neutralize viral infectivity in vitro, the same quantity of fragments that partially protected mice in the form of Fab only slightly delayed virus-induced lethality when injected as scFv fragments, probably because of a much faster in vivo clearance: the biologic half-life was estimated to be about 6 min. Since a scFv derived from a highly neutralizing and protective mAb is only marginally effective in the passive protection of mice from lethal viral infection, the use of such reagents for viral immunotherapy will require strategies to overcome stability limitations.     

γδ T cells are indispensable for interleukin‐23‐mediated protection against Concanavalin A‐induced hepatitis in hepatitis B virus transgenic mice

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation‐induced hepatitis is unknown. In this study, using HBV transgenic (HBs‐Tg) and HBs‐Tg T‐cell receptor‐δ‐deficient (TCR‐δ^−/−) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon‐γ (IFN‐γ) levels and more extensive necrosis. γδ T‐cell deficiency resulted in elevated IFN‐γ in CD4⁺ T cells but not in natural killer or natural killer T cells. The depletion of CD4⁺ T cells and neutralization of IFN‐γ reduced liver damage in HBs‐Tg and HBs‐Tg‐TCR‐δ^−/− mice to a similar extent. Further investigation revealed that HBs‐Tg mice showed an enhanced interleukin‐17 (IL‐17) signature. The administration of exogenous IL‐23 enhanced IL‐17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs‐Tg mice, but not in HBs‐Tg‐TCR‐δ^−/− mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A‐induced hepatitis by inhibiting IFN‐γ production from CD4⁺ T cells and are indispensable for IL‐23‐mediated protection against Con A‐induced hepatitis in HBs‐Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation‐induced liver damage.     

Interferon-γ directly induces gastric epithelial cell death and is required for progression to metaplasia

Chronic inflammation of the gastric mucosa, often caused by autoimmune gastritis and/or infection with Helicobacter pylori, can lead to atrophy of acid-secreting parietal cells with metaplasia of remaining cells. The histological pattern marks a critical step in the progression from chronic gastritis to gastric cancer, yet underlying mechanism(s) of inflammation-induced cell death of gastric epithelial cells are poorly understood. We investigated direct effects of a type 1 cytokine associated with autoimmunity and infection, interferon-γ (IFN-γ), on gastric epithelial cells. IFN-γ was applied to three-dimensional organoid cultures of gastric epithelial cells derived from gastric corpus gland (gastroids) of control and IFN-γ receptor-deficient mice. Gastroids were also treated with supernatants from activated immune cells isolated from a mouse model of autoimmune-mediated atrophic gastritis (TxA23) with and without IFN-γ expression. Finally, histopathological analysis of atrophy and metaplasia severity was performed in TxA23 mice and compared to TxA23 × Ifng^−/− mice. Gastric epithelial cells in gastroid cultures expressed IFN-γ receptor in the basolateral membrane, and gastroids died when treated with IFN-γ in an IFN-γ receptor-dependent manner. Supernatants from immune cells containing high levels of IFN-γ were highly toxic to gastroids, and toxicity was tempered when IFN-γ was either neutralized using a monoclonal antibody or when supernatants from Ifng^−/− mouse immune cells were used. Finally, TxA23 × Ifng^−/− mice showed near-complete abrogation of pre-cancerous histopathological atrophy and metaplasia versus IFN-γ-sufficient controls. We identify IFN-γ as a critical promoter of parietal cell atrophy with metaplasia during the progression of gastritis to gastric atrophy and metaplasia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

IgA is important for clearance and critical for protection from rotavirus infection

Based on a lack of severe phenotype in human immunoglobulin A (IgA) deficiency syndromes, the role of IgA in controlling respiratory and gastrointestinal (GI) infections has not been clearly defined. C57BL/6 and BALB/c mice lacking IgA (IgA^−/−) were developed and used to address this question. When exposed to a common GI virus, rotavirus, IgA^−/− mice exhibited a substantial and significant delay in clearance of the initial infection compared with wild-type mice. IgA^−/− mice excreted rotavirus in stool up to 3 weeks after the initial exposure compared with 10 days observed in wild-type mice. Importantly, IgA^−/− mice failed to develop protective immunity against multiple repeat exposures to the virus. All IgA^−/− mice excreted virus in the stool upon re-exposure to rotavirus, whereas wild-type mice were completely protected against re-infection. These findings clearly indicate a critical role for IgA in the establishment of immunity against a GI viral pathogen.     

Killing Effects of IFN R−/− Mouse NK Cells Activated by HN Protein of NDV on Mouse Hepatoma Cells and Possible Mechanism with Syk and NF-κB

The objective of this article is to evaluate whether the tumoricidal activity of mouse IFN R^−/− nature killer (NK) cells is induced by Newcastle disease virus hemagglutinin-neuraminidase (NDV-HN) stimulation, and to investigate what is the mechanism of the HN-stimulated NK cells to kill mouse hepatoma cell line in vitro. The mouse IFN R^−/− NK cells were stimulated for 16 hr with 500 ng/mL NDV-HN in 1640 medium. Quantify the cytotoxic activities of NK cells against mouse hepatoma cells (Hepa1-6) by flow cytometry. Granzymes B (GrB) and Fas/FasL concentrations in the supernatants of IFN R^−/− NK cells medium were determined by specific ELISA assay. The expression of cell surface GrB and Fas was determined by Western blot. NDV-HN stimulation enhanced tumoricidal activity of IFN R^−/− NK cells toward Hepa1-6 in vitro. Treating with anti-HN neutralizing mAb induced significant decline in the cytotoxicity of IFN R^−/− NK cells toward Hepa1-6 cell line (P < 0.05). After treating with anti-HN protein (1 μL/mL), Syk-specific inhibitor Herbimycin A(250 ng/mL) and NF-κB inhibitor PDTC (500 ng/mL) downregulated the tumoricidal activity of HN-stimulated IFN R^−/− NK cells (P < 0.05). Moreover, significant suppressions in the production of GrB and Fas/FasL were observed in HN-stimulated IFN R^−/− NK cells (P < 0.05). Thus, we concluded that killer activation receptors pathway is involved in the IFN-γ-independent GrB and Fas/FasL expression of NDV-HN-stimulated IFN R^−/− NK cells, and these are activated by Syk and NF-κB. Anat Rec, 302:1718–1725, 2019. © 2019 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association for Anatomy     

IFN-γ induction by neutrophil-derived IL-17A homodimer augments pulmonary antibacterial defense

The role of interleukin-17A (IL-17A) in host defense against Legionella pneumophila remains elusive. To address this issue, we used Il17a^−/−, Il17f^−/−, and Il17a/Il17f^−/− mice on a C57Bl/6 (non-permissive) background and IL-17 neutralizing Abs in mice on an A/J (permissive) background. Higher bacterial (L. pneumophila) counts in the lung and blood along with reduced neutrophil recruitment were detected in Il17a^−/−, but not Il17f^−/−, mice. We found that neutrophils produce IL-17A homodimer (IL-17A) during L. pneumophila infection, and hematopoietic cell-derived IL-17A is known to be important for bacterial clearance. Thus, intratracheal administration of wild-type neutrophils or recombinant IL-17A restored bacterial clearance and neutrophil recruitment in Il17a^−/− mice. Furthermore, neutrophil-depleted Rag2^−/− and Rag2/Il-2rγ^−/− mice exhibited increased bacterial burden, reduced neutrophil influx and IL-17A production in the lung. Recombinant IFN-γ administration in Il17a^−/− mice augmented bacterial elimination, whereas IL-17A administration in Ifnγ^−/− mice did not augment bacterial clearance. IFN-γ is produced by T cells, but not neutrophils or macrophages, suggesting that neutrophil-derived IL-17A induces IFN-γ in a paracrine fashion. Human pneumonic lungs and human neutrophils challenged with L. pneumophila exhibited increased numbers of IL-17A producing cells. These findings display a novel function of neutrophil-derived IL-17A in antibacterial defense via the induction of IFN-γ in a paracrine manner.     

Immunization with Staphylococcus aureus iron regulated surface determinant B (IsdB) confers protection via Th17/IL17 pathway in a murine sepsis model

We have previously shown that IsdB, a conserved protein expressed by Staphylococcus aureus, induces a robust antibody response which correlates with protection in a murine challenge model. Here we investigate the role of cellular immunity in IsdB mediated protection using lymphocyte deficient SCID mice. As opposed to WT CB-17 mice the CB-17 SCID mice were not protected against a lethal challenge of S. aureus after active and passive immunizations with IsdB. Adoptive transfer of in vitro isolated lymphocyte subsets revealed that reconstituting mice with IsdB specific CD3+ or CD4+ T-cells conferred antigen specific protection while CD8 (+) T-cells, CD19 (+) B-cells and plasma cells (CD138 (high) B220 (int) CD19 (lo) ) alone were not protective. A combination of CD3 (+) T-cells plus CD19 (+) B-cells conferred protection in CB-17 SCID mice, whereas bovine serum albumin (BSA) immune lymphocytes did not confer protection. Active immunization experiments indicated that IsdB immunized Jh mice (B-cell deficient) were protected against lethal challenge, while nude (T-cell deficient) mice were not. In vitro assays indicated that isolated IsdB specific splenocytes from immunized mice produced abundant IL-17A, much less IFN-γ and no detectable IL-4. IL-23 deficient mice were not protected from a lethal challenge by IsdB vaccination, pointing to a critical role for CD4 (+) Th17 in IsdB-mediated vaccination. Neutralizing IL-17A, but not IL-22 in vivo significantly increased mortality in IsdB immunized mice; whereas, neutralizing IFN-γ did not alter IsdB-mediated protection. These findings suggest that IL-17A producing Th17 cells play an essential role in IsdB vaccine-mediated defense against invasive S. aureus infection in mice.     

Caspase-11 is expressed in the colonic mucosa and protects against dextran sodium sulfate-induced colitis

Ulcerative colitis and Crohn's disease are major inflammatory syndromes that affect millions of patients. Caspase-11 confers protection against Gram-negative enteropathogens, but its role during colitis is unknown. Here, we show that caspase-11 was constitutively expressed in the colon, and that caspase-11-deficient (caspase-11^−/−) mice were hypersusceptible to dextran sodium sulfate (DSS)-induced colitis. Notably, pro-inflammatory Prevotella species were strongly reduced in the gut microbiota of caspase-11^−/− mice. Co-housing with wild-type mice leveled Prevotella contents, but failed to protect caspase-11^−/− mice from increased susceptibility to DSS-induced colitis. We therefore addressed the role of caspase-11 in immune signaling. DSS-induced tissue damage, release of the pyroptosis/necroptosis marker HMGB1, and inflammatory cell infiltration in the gut were markedly increased in caspase-11^−/− mice. Moreover, caspase-11^−/− mice showed normal or increased production of mature interleukin (IL)-1β and IL-18, whereas IL-1β and IL-18 secretion was blunted in animals lacking both caspases 1 and 11. In conclusion, we showed that caspase-11 shapes the gut microbiota composition, and that caspase-11^−/− mice are highly susceptible to DSS-induced colitis. Moreover, DSS-induced inflammasome activation relied on caspase-1, but not caspase-11. These results suggest a role for other caspase-11 effector mechanisms such as pyroptosis in protection against intestinal inflammation.     

Protection of Batf3-deficient mice from experimental cerebral malaria correlates with impaired cytotoxic T-cell responses and immune regulation

Excessive inflammatory immune responses during infections with Plasmodium parasites are responsible for severe complications such as cerebral malaria (CM) that can be studied experimentally in mice. Dendritic cells (DCs) activate cytotoxic CD8⁺ T-cells and initiate immune responses against the parasites. Batf3^−/− mice lack a DC subset, which efficiently induces strong CD8 T-cell responses by cross-presentation of exogenous antigens. Here we show that Batf3^−/− mice infected with Plasmodium berghei ANKA (PbA) were protected from experimental CM (ECM), characterized by a stable blood−brain barrier (BBB) and significantly less infiltrated peripheral immune cells in the brain. Importantly, the absence of ECM in Batf3^−/− mice correlated with attenuated responses of cytotoxic T-cells, as their parasite-specific lytic activity as well as the production of interferon gamma and granzyme B were significantly decreased. Remarkably, spleens of ECM-protected Batf3^−/− mice had elevated levels of regulatory immune cells and interleukin 10. Thus, protection from ECM in PbA-infected Batf3^−/− mice was associated with the absence of strong CD8⁺ T-cell activity and induction of immunoregulatory mediators and cells.     

Intestinal IFN-γ production is associated with protection from clinical signs,but not with elimination of worms,in Echinostoma caproni infected-mice

In the present paper, we assess the relationship between the expression of IFN-γ and the development of clinical signs in Echinostoma caproni-infected mice. For this purpose, we studied the course of the infection in three mouse strains: ICR (CD-1®) (a host of high compatibility with E. caproni), BALB/c (a prototypical Th2 strain), and BALB/c deficient for IFN-γ mice (IFN-γ^?/?). Infection in ICR mice is characterized by the elevated expression of IFN-γ and iNOS in the intestine concomitantly with the lack of clinical signs. In contrast, the infection was more virulent in BALB/c and IFN-γ-deficient mice that developed a severe form of the disease together with the absence of IFN-γ expression. The disease was more severe in IFNγ^?/? mice in which the disease was lethal during the few first weeks of the infection. The analysis of different parameters of the infection in each host strain showed that most of the features were similar in the three mouse strains, suggesting the IFN-γ plays a central role in that protection against severe disease. Thus, IFN-γ seems to play a dichotomous role in the infection facilitating the parasite establishment, but it may also benefit mice since it protects the mice from morbidity and mortality induced by the parasite.     

Pure CD4+ T lymphocytes fail to protect perorally infected SCID mice from lethal microsporidiosis caused by Encephalitozoon cuniculi

The role of CD4+ and CD8+ T lymphocytes in the protection against intraperitoneally (i.p.) or perorally (p.o.) acquired Encephalitozoon cuniculi (Levaditi et al., C R Soc Biol Paris 89:984–986, 1923) infection was studied by means of reconstitution of severe combined immunodeficiency (SCID) mice with well-defined populations of naive CD8+ or CD4+ T lymphocytes. Adoptive transfer of pure CD8+ T lymphocyte subpopulation protects SCID mice against a lethal microsporidiosis caused by E. cuniculi. The protective effect of CD8+ T lymphocytes is manifested in both i.p. and p.o. infection. On the contrary, the host defense against peroral infection does not require CD8+ T cells. The protective role is not mediated by CD4+ T lymphocytes only. SCID mice reconstitution with pure CD4+ T cell subpopulation led to prolonged survival of perorally infected mice. However, these mice died due to lethal encephalitozoonosis caused by i.p. infection.     

The medulla controls effector primed γδT-cell development in the adult mouse thymus

γδT cells are produced in the thymus throughout life and provide immunity at epithelial-rich sites. Unlike conventional αβT cells, γδT-cell development involves intrathymic acquisition of effector function, with priming for either IL17 or IFN-γ production occurring during embryonic or adult life, respectively. How the thymus controls effector-primed γδT-cell generation in adulthood is poorly understood. Here, we distinguished de novo γδT cells from those undergoing thymus recirculation and/or retention using Rag2GFP mice alongside markers of maturation/effector priming including CD24, CD25, CD73, and IFN-γ, the latter by crossing with IFN-γ^YFP GREAT mice. We categorize newly developing γδT-cells into an ordered sequence where CD25⁺CD73⁻IFN-γ^YFP− precursors are followed sequentially by CD25⁻CD73⁺IFN-γ^YFP− intermediates and CD25⁻CD73⁺IFN-γ^YFP+ effectors. To determine intrathymic requirements controlling this sequence, we examined γδT-cell development in Relb^−/− thymus grafts that lack medullary microenvironments. Interestingly, medulla deficiency did not alter CD25⁺ γδT-cell precursor generation, but significantly impaired development of effector primed stages. This impact on γδT-cell priming was mirrored in plt/plt mice lacking the medullary chemoattractants CCL19 and CCL21, and also Ccl21a^−/− but not Ccl19^−/− mice. Collectively, we identify the medulla as an important site for effector priming during adult γδT-cell development and demonstrate a specific role for the medullary epithelial product CCL21 in this process.     

CCR2 mediates increased susceptibility to post-H1N1 bacterial pneumonia by limiting dendritic cell induction of IL-17

Post influenza bacterial pneumonia is associated with significant mortality and morbidity. Dendritic cells (DCs) play a crucial role in host defense against bacterial pneumonia, but their contribution to post influenza-susceptibility to secondary bacterial pneumonia is incompletely understood. WT and CCR2^−/− mice were infected with 100 plaque forming units (pfu) H1N1 intranasally alone or were challenged on day 5 with 7 × 10⁷ colony forming units (cfu) methicillin-resistant Staphylococcus aureus intratracheally. WT mice express abundant CCL2 mRNA and protein post-H1N1 alone or dual infection. CCR2^−/− mice had significantly higher survival as compared to WT mice, associated with significantly improved bacterial clearance at 24 and 48 h (10-fold and 14-fold, respectively) post bacterial challenge. There was robust upregulation of IL-23 and IL-17 as well as downregulation of IL-27 expression in CCR2^−/− mice following sequential infection as compared to WT mice, which was also associated with significantly greater accumulation of CD103⁺ DC. Finally, WT mice treated with a CCR2 inhibitor showed improved bacterial clearance in association with similar cytokine profiles as CCR2^−/− mice. Thus, CCR2 significantly contributes to increased susceptibility to bacterial infection after influenza pneumonia likely via altered dendritic cell responses and thus, CCR2 antagonism represents a potential therapeutic strategy.     

Interleukin-17/interleukin-17 receptor axis elicits intestinal neutrophil migration,restrains gut dysbiosis and lipopolysaccharide translocation in high-fat diet-induced metabolic syndrome model

Sound evidence supports a role for interleukin-17 (IL-17) -producing γδ T cells and IL-17-producing helper T (Th17) cells in intestinal homeostasis, especially in intestinal barrier integrity. In the present study, we aimed to evaluate the role of IL-17 cytokine in the regulation of intestinal immunity and obesity-induced metabolic syndrome (MetS) in an experimental murine model. C57BL/6 wild-type (WT) mice and mice lacking the IL-17 cytokine receptor (IL-17RA^−/−) were fed either a control diet (CD) or a high-fat diet (HFD) for 9 weeks. Our data demonstrate that IL-17RA^−/− mice are protected against obesity, but develop hyperglycemia, hyperinsulinemia and insulin resistance. In parallel, HFD-fed IL-17RA^−/− mice display intense inflammation in the ileum compared with WT mice on the HFD. IL-17RA^−/− mice fed the HFD exhibit impaired neutrophil migration to the intestinal mucosa and reduced gene expression of the CXCL-1 chemokine and CXCR-2 receptor in the ileum. Interestingly, the populations of neutrophils (CD11b⁺ Ly6G⁺) and anti-inflammatory macrophages (CD11b⁺ CX3CR1⁺) are increased in the mesenteric lymph nodes of these mice. IL-17RA^−/− mice on the HFD also display increased commensal bacterial translocation into the bloodstream and elevated lipopolysaccharide (LPS) levels in the visceral adipose tissue (VAT). Metagenomic analysis of bacterial 16S gene revealed increased Proteobacteria and Bacteroidetes phyla, the main representatives of Gram-negative bacteria, and reduced Akkermansia muciniphila in the fecal samples of IL-17RA^−/− mice fed the HFD. Together, these data indicate that the IL-17/IL-17R axis drives intestinal neutrophil migration, limits gut dysbiosis and attenuates LPS translocation to VAT, resulting in protection to MetS.     

Immunity to viruses in B cell-deficient mice: Influence of antibodies on virus persistence and on T cell memory

Mice rendered B cell deficient by targeted disruption of the immunoglobulin μ chain gene (IgM−/− mice) were used to analyze the role of antibodies and B cells in viral infections; homozygous IgM−/− mice were bred in a way to avoid transmission of maternal antibodies. After infection with vesicular stomatitis virus (VSV), IgM−/− mice developed paralytic disease and subsequently died, whereas C57BL/6 control mice or IgM−/− mice passively protected with VSV-neutralizing antibodies survived. Furthermore, IgM−/− mice showed increased natural killer (NK) activity upon exposure to either lymphocytic choriomeningitis virus (LCMV) or to poly(I) · poly(C), while NK activity in untreated IgM−/− mice was within normal ranges. Cytotoxic T cell responses were comparable in IgM−/− and control mice infected either with VSV or with vaccinia virus or with low doses of LCMV (10² infectious focus-forming units [ifu]). After intracerebral infection with LCMV-Armstrong, CD8⁺ T cell-mediated lethal lymphocytic choriomeningitis developed independently of the presence of B cells and antibodies. After infection with high doses (2 × 10⁶ − 5 × 10⁶ ifu) of LCMV-WE or LCMV-Docile, IgM−/− mice exhibited a reduced capacity to control these primary infections and had elevated virus liters for prolonged times (>60 days). Nevertheless, the cytotoxic T cell response against LCMV in the early phase of infection was comparable in IgM−/− and control mice, but disappeared in those IgM−/− mice which had a persistent viral infection. Cytotoxic T cell memory was apparently unimpaired in low-dose-primed IgM−/−mice, which were able to control the primary virus infection; both IgM−/− and control mice cleared a high intravenous dose of virus within 2 days after challenge infection. This indicates that an efficient T cell memory against LCMV was established in the absence of B cells.     

Protective capacity and epitope specificity of CD8(+) T cells responding to lethal West Nile virus infection

West Nile virus (WNV) is a small, positive‐strand RNA virus belonging to the Flaviviridae genus, which causes lethal encephalitis in a subset of infected birds and mammals. In humans, WNV exhibits pronounced age‐related morbidity and mortality, but the basis of this effect is unclear, and the molecular and cellular parameters of the host‐WNV infection are just beginning to be elucidated. Indeed, numerous mechanisms were implicated in protection in vivo against WNV (IFN‐I and IFN‐γ, antibody, C’, CD8 and CD4 T cells), but the individual importance of each one of these remains unclear. Here, we show that transfer of highly enriched naïve CD8⁺ T cells protects the majority of alymphoid mice against lethal WNV infection. To substantiate and expand this finding, we defined the peptide specificity of the CD8 response in H‐2b mice and used a panel of identified peptides to map one dominant (NS4b _2248–2256) and several subdominant epitopes. The hierarchy of these epitopes was stably maintained in the memory responses. Most importantly, CTL lines directed against these peptides conferred protection against lethal WNV infection in direct proportion to the epitope immunodominance. These results provide a springboard for future characterization of T cell responses against WNV and demonstrate, for the first time, that CD8 T cells can single‐handedly protect from this disease.     

Blocking by anti-idiotypic antibodies of monoclonal antibody mediated protection in mice against encephalomyocarditis virus induced diabetes and lethal disease

Summary Monoclonal antibodies (MA) UM 21.1 and UM 21.2 protect mice against encephalomyocarditis virus (D-variant) induced diabetes and lethal disease. MA-mediated protection in vivo as well as neutralization in vitro could be specifically blocked by anti-idiotypic antibodies.