Background The delayed-type hypersensitivity arthritis (DTHA) model combines collagen type II antibody (anti-CII) treatment with local DTH induction in the footpad [1] which induces a strong arthritis accompanied by bone destruction and excessive peri-articular bone formation. Objectives Here we analyzed the impact of osteoclasts on inflammation, joint damage and bone formation in the DTHA model. Methods C57BL/6 mice were immunized with methylated BSA (mBSA) in CFA, given 1000 μg anti-CII four days later [1] and challenged seven days after immunization with 200 μg mBSA in PBS in one hind foot pad and with 20 μl PBS only in the other foot pad (control). Osteoclast activity was determined in vivo by fluorescence imaging using the Cat K 680 FAST probe. Receptor-activator of nuclear factor kappa B (RANKL), TRAP and Cathespin K (CatK) mRNA expression was analyzed in arthritic paw tissue by mRNA deep sequencing. TRAP5b, CTX-I and CTX-II serum levels were determined by ELISA. In situ expression of tartrate-resistant acid phosphatase (TRAP), collagen 10 (COL10) and osteocalcin was detected by immunohistochemical staining. Inflammation, erosive joint damage and peri-articular bone formation were semi-quantitatively scored. Results In vivo imaging of osteoclast activity showed increased CatK activity on days 3 and 8 after arthritis induction in the arthritic paw. RANKL mRNA expression peaked on d3 while TRAP and CatK mRNA expression peaked on d8. Erosive joint damage was driven from the subchrondal bone marrow side. Here, TRAP-positive osteoclasts were located at the borders of a granulation tissue which eroded the subchondral bone plate. Peri-articular bone formation involved COL10 expressing hypertrophic chondrocytes as well as osteocalcin–expressing osteoblasts. Treatment with anti-RANKL antibody did not affect footpad swelling (p>0.05) but reduced the serum levels of TRAP5b (p<0.001) and CTX-I (p<0.001) and prevented the destruction of the subchondral bone plate (p<0.001) and development of the invasive granulation tissue (p<0.001) at subchondral sites. Scores of intra- and peri-articular inflammation and scores of peri-articular bone formation did not change (p>0.05). Conclusions Inhibition of osteoclast differentiation prevents joint destruction in the DTHA model driven by the subchondral granulation tissue while leaving intra-articular and peri-articular inflammation and bone formation unaffected. Osteoclast activity might be targeted to prevent erosive damage driven from subchondral sites as for instance in sacroiliacal joints of patients with spondyloarthritis. Disclosure of Interest None declared

Background C5a is an anaphylatoxin generated in response to complement activation. C5a promotes leukocyte chemotaxis and activation via the C5a receptor (C5aR), both directly on C5aR-positive cells such as neutrophils, monocytes and macrophages, and indirectly on T cells via effects on C5aR-positive antigen specific cells (APCs)1. C5a production and C5aR signalling have been implicated in a wide range of inflammatory disorders including rheumatoid arthritis (RA)2. DTH arthritis is a single-paw arthritis model, which is dependent on CD4+ T cells. Besides T cells, neutrophils and macrophages are involved in the pathology, and inflammatory mediators associated with these cells are induced locally in the arthritic paw3 Objectives The purpose of this study was to investigate the therapeutic effect of a blocking anti-C5aR antibody in a single–paw arthritis model in mice in both a 2-week multiple-dose (MD) study and in a single-dose (SD) study with read-out at 60 h post-dosing. Methods DTH arthritis was induced as described by Atkinson3. In the MD study mice were treated twice weekly with anti-C5aR antibody (25 mg/kg/dose) from the day of arthritis induction. Mechanistic studies were conducted to investigate the immune-modulating effect of a SD of 25 mg/kg anti-C5aR with read-out at 60 h post-dosing/arthritis induction. Cytokine/chemokine levels in whole–paw homogenate supernatants were analyzed using ELISA and bioplex. Paw sections were evaluated histologically, and composition of leukocyte subsets isolated from lymph nodes and arthritic paws were analyzed by flow cytometry. Statistics: unpaired two-sided t-test. Results Significant reduction in paw swelling (P<0.01) and histopathology score (P<0.0001) was observed in the MDstudy. Swelling of the arthritic paw was also significantly reduced compared to control–treated mice in the SD study (P<0.0001). Also a single dose of anti-C5aR resulted in significantly reduced levels of MIP-2, MCP-1 and TNFα (P<0.05 for each analyte) in the arthritic paw compared to the levels found in control-treated mice. However, no difference was detected in the total number of leukocytes infiltrating the arthritic paws 60 h after SD treatment. Flow cytometric analysis of leukocyte subsets isolated from arthritic pawsafter SD treatment revealed significant reductions in CD4+ T cell counts of anti-C5aR-treated mice compared to control-treated mice (P<0.05), which correlated with significantly reduced CD4+ T cell proliferation (P<0.05) in the draining popliteal lymph node. Target validation in control-treated DTH arthritic mice revealed expression of C5aR on neutrophils (blood and paw) and on a subset of macrophages (paw), but not on CD4+ T cells. Conclusions Our studies reveal a significant effect of anti-C5aR treatment on DTH arthritis disease activity, and our mechanistic SD studies strongly suggests that the effect is mediated via reduced local production of inflammatory mediators and reduced CD4+ T cell proliferation, while no effect on leukocyte chemotaxis was observed 60 h after SD anti-C5aR antibody treatment. The effect on CD4+ T cell proliferation is probably indirect via effects of an anti-C5aR antibody on APCs. References Dunkelberger JR et al.(2010) Mol Immunol 47: 2176–2186; Lee H et al. (2008) Immunol Cell Biol 86: 153–160; Atkinson SA et al. (2012) Arthritis Res Ther 14(3):R134 Disclosure of Interest A. Nansen Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, S. Atkinson Employee of: PhD student, Research performed at Novo Nordisk A/S, P. Usher Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, C. Haase Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S, L. Hornum Shareholder of: Novo Nordisk A/S, Employee of: Novo Nordisk A/S

T cells play a key role in the control of viral infection in the CNS but may also contribute to immune-mediated cell damage. To study the redundancy of the chemokine receptors CXCR3 and CCR5 in regulating virus-induced CD8+ T cell-mediated inflammation in the brain, CXCR3/CCR5 double-deficient mice were generated and infected intracerebrally with noncytolytic lymphocytic choriomeningitis virus. Because these chemokine receptors are mostly expressed by overlapping subsets of activated CD8+ T cells, it was expected that absence of both receptors would synergistically impair effector T cell invasion and therefore protect mice against the otherwise fatal CD8+ T cell-mediated immune attack. Contrary to expectations, the accumulation of mononuclear cells in cerebrospinal fluid was only slightly delayed compared with mice with normal expression of both receptors. Even more surprising, CXCR3/CCR5 double-deficient mice were more susceptible to intracerebral infection than CXCR3-deficient mice. Analysis of effector T cell generation revealed an accelerated antiviral CD8+ T cell response in CXCR3/CCR5 double-deficient mice. Furthermore, while the accumulation of CD8+ T cells in the neural parenchyma was significantly delayed in both CXCR3- and CXCR3/CCR5-deficient mice, more CD8+ T cells were found in the parenchyma of double-deficient mice when these were analyzed around the time when the difference in clinical outcome becomes manifest. Taken together, these results indicate that while CXCR3 plays an important role in controlling CNS inflammation, other receptors but not CCR5 also contribute significantly. Additionally, our results suggest that CCR5 primarily functions as a negative regulator of the antiviral CD8+ T cell response.

T cells play an important role in controlling viral infections inside CNS. To study the role of the chemokine receptor CXCR3 in the migration and positioning of virus-specific effector T cells within the brain, CXCR3 deficient mice were infected intracerebrally (i.e.) with lymphocytic choriomeningitis virus (LCMV). Most CXCR3 deficient mice survived the infection, whereas wildtype(WT) mice died from CD8(+) T-cell mediated immunopathology. However, reconstitution of CXCR3 deficient mice with WT CF8(+) T cells restored susceptibility to LCMV-induced meningitis. Immunohistological analysis showed a striking impairment of CXCR3 deficient CD8' T cells to migrate from the meninges into the outer layers of the brain parenchyma. Thus, CXCR3 seems to play a critical role in the positioning of effector T cells at sites of viral inflammation in the brain.

T-cells play an important role in controlling viral infections inside the CNS. To study the role of the chemokine receptor CXCR3 in the migration and positioning of virus-specific effector T-cells within the brain, CXCR3-deficient mice were infected intracerebrally with lymphocytic choriomeningitis virus (LCMV). Analysis of the induction phase of the antiviral CD8 + T-cell response did not reveal any immune defects in CXCR3-deficient mice. Yet, when mice were challenged with LCMV intracerebrally, most CXCR3-deficient mice survived the infection, whereas wild-type mice invariably died from CD8 + T-cell-mediated immunopathology. Quantitative analysis of the cellular infiltrate in CSF of infected mice revealed modest, if any, decrease in the number of mononuclear cells recruited to the meninges in the absence of CXCR3. However, immunohistological analysis disclosed a striking impairment of CD8 + T-cells from CXCR3-deficient mice to migrate from the meninges into the outer layers of the brain parenchyma despite similar localization of virus-infected target cells. Reconstitution of CXCR3-deficient mice with wild-type CD8 + T-cells completely restored susceptibility to LCMV-induced meningitis. Thus, taken together, our results strongly point to a critical role for CXCR3 in the positioning of effector T-cells at sites of viral inflammation in the brain.

ABSTRACTThe immune response to lymphocytic choriomeningitis virus in mice lacking macrophage inflammatory protein-1α (MIP-1α) was evaluated. Generation of virus-specific effector T cells is unimpaired in MIP-1α-deficient mice. Furthermore, MIP-1α is not required for T-cell-mediated virus control or virus-induced T-cell-dependent inflammation. Thus, MIP-1α is not mandatory for T-cell-mediated antiviral immunity.

T cell mediated immunity and in particular CD8+ T cells are pivotal for the control of most viral infections. T cells exclusively exert their antiviral effect through close cellular interaction with relevant virus-infected target cells in vivo. It is therefore imperative that efficient mechanisms exist, which will rapidly direct newly generated effector T cells to sites of viral replication. In the present report we have reviewed our present knowledge concerning the molecular interactions, which are important in targeting of effector CD8+ T cells to sites of viral infection.

ABSTRACT The human herpesvirus 8-encoded protein vMIP-II is a potent in vitro antagonist of many chemokine receptors believed to be associated with attraction of T cells with a type 1 cytokine profile. For the present report we have studied the in vivo potential of this viral chemokine antagonist to inhibit virus-induced T-cell-mediated inflammation. This was done by use of the well-established model system murine lymphocytic choriomeningitis virus infection. Mice were infected in the footpad, and the induced CD8+ T-cell-dependent inflammation was evaluated in mice subjected to treatment with vMIP-II. We found that inflammation was markedly inhibited in mice treated during the efferent phase of the antiviral immune response. In vitro studies revealed that vMIP-II inhibited chemokine-induced migration of activated CD8+ T cells, but not T-cell-target cell contact, granule exocytosis, or cytokine release. Consistent with these in vitro findings treatment with vMIP-II inhibited the adoptive transfer of a virus-specific delayed-type hypersensitivity response in vivo, but only when antigen-primed donor cells were transferred via the intravenous route and required to migrate actively, not when the cells were injected directly into the test site. In contrast to the marked inhibition of the effector phase, the presence of vMIP-II during the afferent phase of the immune response did not result in significant suppression of virus-induced inflammation. Taken together, these results indicate that chemokine-induced signals are pivotal in directing antiviral effector cells toward virus-infected organ sites and that vMIP-II is a potent inhibitor of type 1 T-cell-mediated inflammation.

The CC chemokine receptor CCR5 is an important coreceptor for human immunodeficiency virus (HIV), and there is a major thrust to develop anti-CCR5-based therapies for HIV-1. However, it is not known whether CCR5 is critical for a normal antiviral T-cell response. This study investigated the immune response to lymphocytic choriomeningitis virus in mice lacking CCR5 (CCR5(-/-) mice). This infection is a classical model for studying antiviral immunity, and influx of CCR5-expressing CD8(+) T cells and macrophages is essential for both virus control and associated immunopathology. Results showed that the virus-induced clonal expansion of antigen-specific T cells was augmented in CCR5(-/-) mice especially with regard to the CD4(+) subset. Despite absence of CCR5, intracerebral infection invariably resulted in lethal T cell-mediated meningitis, and quantitative and qualitative analysis of the inflammatory exudate cells did not reveal any significant differences between gene-targeted mice and wild-type controls. CCR5 was also found to be redundant regarding the ability to eliminate virus from internal organs. Using delayed-type hypersensitivity to evaluate CD8(+) T cell-mediated inflammation, no significant influence of CCR5 was found, not even when viral peptide was used as local trigger instead of live virus. Finally, long-term CD8(+) T cell-mediated immune surveillance was efficiently sustained in CCR5(-/-) mice. Taken together, these results indicate that expression of CCR5 is not critical for T cell-mediated antiviral immunity, and this molecule may therefore constitute a logic and safe target for anti-HIV therapies.

LPS is the major active agent in the pathogenesis of Gram-negative septic shock. In this report we have studied the influence of concurrent viral infection on the outcome of LPS-induced shock. We find that infection with vesicular stomatitis virus sensitizes mice to LPS at an early time point following infection. Treatment of mice with the chemical IFN inducer, polyinosinic:polycytidylic acid, has a similar effect. This hypersensitivity to LPS correlated with hyperproduction of TNF-alpha in vivo. The cellular and molecular mechanisms underlying this phenomenon were investigated using Ab-depleted and gene-targeted mice. Our results revealed that while NK cell depletion and elimination of IFN-gamma partially protected against the sensitizing effects of vesicular stomatitis virus and polyinosinic:polycytidylic acid, the most striking effect was observed in IFN-alphabetaR-deficient mice. Thus hyperproduction of TNF-alpha was completely abrogated in IFN-alphabetaR-deficient mice, indicating that the principal mechanism underlying rapid virus-induced sensitization to LPS is an IFN-alphabeta-mediated priming of mice for an augmented production of TNF-alpha in response to LPS. This conclusion was further supported by the finding that pretreatment of mice with rIFN-alphabeta mimicked the effect of viral infection. In conclusion, our results reveal a previously unrecognized proinflammatory effect of IFN-alphabeta and point to a new pathway through which viral infection may influence the outcome of concurrent bacterial infection.

With the aim of characterizing the antiviral immune response to a non-cytocidal virus, we studied the outcome of lymphocytic choriomeningitis virus infection in a number of gene knockout mouse strains. Two virus strains differing markedly in their capacity to spread and replicate inside the murine host were used. Our results reveal that very different outcomes may be observed depending on virus strain and immunocompetence of the host. Thus while CD4+ cells are not critical during the initial phase of virus control, infectious virus reappear in mice lacking CD4+ cells, B cells or CD40 ligand. Reappearance of virus is associated with impaired long-term CD8+ T-cell mediated immune surveillance, and the time to virus resurgence is inversely correlated to the replication rate of the virus. Our studies also reveal that interferon-gamma is a central cytokine, and depending on the rate of virus replication, mice lacking the ability to produce interferon-gamma may develop either a severe, mostly fatal, T-cell mediated wasting syndrome or a chronic infection characterized by long-term coexistence of antiviral cytotoxic T lymphocytes and infectious virus. Mathematical modelling indicates that these different outcomes may be explained in relatively simple mathematical terms. This suggests that modelling may be used as a means to predict critical host and virus parameters. Therefore, combining mathematical modelling with precise, quantitative, in vivo analyses looks to be a promising approach in addressing central quantitative issues in immunobiology.

High-affinity IgG autoantibodies (aAb) to IL-1alpha are among the most frequently found aAb to cytokines in humans. To establish an animal model with aAb to IL-1alpha, we immunised mice with recombinant murine IL-1alpha. Unprimed and Bacille Calmette-Guérin (BCG)-primed BALB/cA mice were vaccinated with IL-1alpha coupled to purified protein derivative of tuberculin (PPD). Both unprimed and primed animals developed IgG aAb to IL-1alpha. These aAb persisted at high levels more than 100 days after vaccination and did not cross-react with murine IL-1beta. The induced anti-IL-1alpha aAb inhibited binding of IL-1alpha to the murine T-cell line NOB-1 by simple competition and neutralised IL-1alpha, but not IL-1beta-induced IL-6 in vivo. The aAb did not induce visible discomfort in the animals. In conclusion, long-lasting and high levels of neutralising and specific IgG aAb to IL-1alpha can be induced in mice by vaccination with recombinant murine IL-1alpha conjugated to PPD. Studies of the effects of IL-1alpha aAb in such animals may help clarify the importance of naturally occurring IL-1alpha aAb in humans and permit the evaluation of future therapies with cytokine aAb in patients with immunoinflammatory diseases and cytokine-dependent tumours.

Chemokines and their receptors play a critical role in the selective recruitment of various leukocyte subsets. In this study, we correlated the expression of multiple chemokine and CC chemokine receptor (CCR) genes during the course of intracerebral (i.c.) infection with lymphocytic choriomeningitis virus (LCMV) and vesicular stomatitis virus (VSV), which are prototypic of a noncytopathic and a cytopathic virus, respectively. Infection of mice with either virus resulted in rapid activation and overlapping cerebral expression of a number of chemokine genes. Infection with VSV i.c. causes a rapidly lethal, T cell-independent encephalitis, and infection resulted in a dramatic early up-regulation of chemokine gene expression. Similar marked up-regulation of chemokine expression was not seen until late after LCMV infection and required the presence of activated T cells. Cerebral CCR gene expression was dominated by CCR1, CCR2 and CCR5. However, despite a stronger initial chemokine signal in VSV-infected mice, only LCMV-induced T cell-dependent inflammation was found to be associated with substantially increased expression of CCR genes. Virus-activated CD8+ T cells were found to express CCR2 and CCR5, whereas activated monocytes/macrophages expressed CCR1 in addition to CCR2 and CCR5. Together, these CCR profiles readily account for the CCR profile prominent during CD8+-dependent CNS inflammation.

The role of soluble receptors for TNF-alpha (sTNF-Rs) as markers of virus-induced host responses was studied by the use of murine model infections. A marked elevation in serum levels of sTNF-R75, but not sTNF-R55, was found 1 day after infection with vesicular stomatitis virus (VSV). In mice infected with lymphocytic choriomeningitis virus (LCMV), an early increase was also revealed, but peak levels of sTNF-R75 were observed later temporally related to maximal T cell-mediated anti-viral activity. Analysing different well characterized knockout mice, it was found that elevated release of sTNF-R75 into serum early after VSV infection was independent of T cells, whereas interferon (IFN)-alpha/beta seemed to be a major mediator. In contrast, increased release of sTNF-R75 into serum 8 days post-LCMV infection was mediated via T cells but independently of both CD40 ligand and IFN-gamma. A simple correlation between release of sTNF-Rs in vivo and macrophage activation in vitro was not present. These findings indicate that sTNF-R75 is indeed a sensitive marker of both innate and specific cell-mediated host reactivity during viral infection, but it is not correlated to a single immunological parameter.

To investigate the mechanism(s) whereby T cells protect against a lethal outcome of systemic infection with vesicular stomatitis virus, mice with targeted defects in genes central to T cell function were tested for resistance to i.v. infection with this virus. Our results show that mice lacking the capacity to secrete both IFN-gamma and perforin completely resisted disease. Similar results were obtained using IL-4 knockout mice, indicating that neither cell-mediated nor T(h)2-dependent effector systems were required. In contrast, mice deficient in expression of CD40 ligand were more susceptible than wild-type mice, and residual resistance in these mice was almost completely abrogated by depletion of CD8(+) T cells. In keeping with this, mice lacking both MHC class I and class II expression succumbed to the infection, whereas most class II-deficient mice normally survive. Adoptive transfer experiments using B cell- and T cell-deficient recipients revealed that no protection could be obtained in the absence of B cells, whereas treatment with virus-specific immune (IgG) serum controlled viral spreading to the central nervous system (CNS), but did not necessarily accomplish virus elimination. Taken together, these results underscore that B cells are essential in preventing early infection of the CNS, but T cells are required for long-term survival. CD4(+) T cells are most efficient in this context and the key function is to provide cognate help to B cells. However, if CD4(+) cell function is compromised, CD8(+) T cells become critical and may suffice for survival.

By using mice with a targetted disruption in the gene encoding inducible nitric-oxide synthase (iNOS), we have studied the role of nitric oxide (NO) in lymphocytic choriomeningitis virus (LCMV)-induced, T cell-mediated protective immunity and immunopathology. The afferent phase of the T cell-mediated immune response was found to be unaltered in iNOS-deficient mice compared with wild-type C57BL/6 mice, and LCMV- induced general immunosuppression was equally pronounced in both strains. In vivo analysis revealed identical kinetics of virus clearance, as well as unaltered clinical severity of systemic LCMV infection in both strains. Concerning the outcome of intracerebral infection, no significant differences were found between iNOS-deficient and wild-type mice in the number or composition of mononuclear cells found in the cerebrospinal fluid on day 6 post-infection. Likewise, NO did not influence the up-regulation of proinflammatory cytokine/chemokine genes significantly, nor did it influence the development of fatal meningitis. However, a reduced virus-specific delayed-type hypersensitivity reaction was observed in iNOS-deficient mice compared with both IFN-gamma-deficient and wild-type mice. This might suggest a role of NO in regulating vascular reactivity in the context of T cell-mediated inflammation. In conclusion, these findings indicate a minimal role for iNOS/NO in the host response to LCMV. Except for a reduced local oedema in the knockout mice, iNOS/NO seems to be redundant in controlling both the afferent and efferent phases of the T cell-mediated immune response to LCMV infection.

CD40 ligand (CD40L) is an important molecule that is known to be involved in T-B collaboration and certain aspects of cell-mediated immunity. However, its role in antiviral immunity has not been clearly defined as of yet. Therefore, mice with a targeted defect in the gene encoding this molecule were infected with one of two strains of lymphocytic choriomeningitis virus differing markedly in their capacity to spread in the host. Infection with lymphocytic choriomeningitis virus is initially controlled primarily by CD8+ effector cells, whereas long-term immune surveillance also depends upon CD4+ cells and B cells. Our results reveal that the primary activation, clonal expansion, and differentiation of CD8+ T cells does not require expression of CD40L. However, lack of expression results in rapid impairment of CTL responsiveness and failure to permanently control virus replication. This happens not only in mice infected with the rapidly spreading virus strain but also at a late stage in mice infected with the strain of more limited potential for spreading. In the latter mice, virus replication is initially controlled very efficiently, but high levels of virus can be detected in the blood and internal organs approximately 6 mo after virus inoculation. Since the impairment of immune function seems to be more pronounced in CD40L-deficient mice than in mice lacking either CD4+ cells or B cells, these results indicate that CD40L is pivotal to sustain efficient antiviral immune surveillance, including CD8+ T cells, and suggest that CD40L is critically involved in cellular interactions in addition to T-B cooperation.

Effector T cells constitute a critical component in the immune response to most viral infections (Doherty et al. 1992). Consequently, understanding the mechanisms regulating the generation and function of effector T cells is central to the study of viral pathogenesis. It is characteristic that T cells — as opposed to B cells — are only able to exert their effector function within a very limited distance. Therefore, once effector T cells are generated, they must be able to migrate to relevant sites of infection. This requires a set of surface receptors which direct the migration of effector cells to infected areas. However, not only must fully differentiated effector T cells be able to reach any part of the organism, but, it is also nescessary for naive T cells to continually recirculate in order for the immune system to optimally utilize the limited number of cells with T cell receptors (TCRs) relevant to a given antigen.