Modulation of Monocyte-Driven Myositis in Alphavirus Infection Reveals a Role for CX3CR1+ Macrophages in Tissue Repair.

Arthritogenic alphaviruses such as Ross River and Chikungunya viruses cause debilitating muscle and joint pain and pose significant challenges in the light of recent outbreaks. How host immune responses are orchestrated after alphaviral infections and lead to musculoskeletal inflammation remains poorly understood. Here, we show that myositis induced by Ross River virus (RRV) infection is driven by CD11bhi Ly6Chi inflammatory monocytes and followed by the establishment of a CD11bhi Ly6Clo CX3CR1+ macrophage population in the muscle upon recovery. Selective modulation of CD11bhi Ly6Chi monocyte migration to infected muscle using immune-modifying microparticles (IMP) reduced disease score, tissue damage, and inflammation and promoted the accumulation of CX3CR1+ macrophages, enhancing recovery and resolution. Here, we detail the role of immune pathology, describing a poorly characterized muscle macrophage subset as part of the dynamics of alphavirus-induced myositis and tissue recovery and identify IMP as an effective immunomodulatory approach. Given the lack of specific treatments available for alphavirus-induced pathologies, this study highlights a therapeutic potential for simple immune modulation by IMP in infected individuals in the event of large alphavirus outbreaks.IMPORTANCE Arthritogenic alphaviruses cause debilitating inflammatory disease, and current therapies are restricted to palliative approaches. Here, we show that following monocyte-driven muscle inflammation, tissue recovery is associated with the accumulation of CX3CR1+ macrophages in the muscle. Modulating inflammatory monocyte infiltration using immune-modifying microparticles (IMP) reduced tissue damage and inflammation and enhanced the formation of tissue repair-associated CX3CR1+ macrophages in the muscle. This shows that modulating key effectors of viral inflammation using microparticles can alter the outcome of disease by facilitating the accumulation of macrophage subsets associated with tissue repair.

Effects of an In-Frame Deletion of the 6k Gene Locus from the Genome of Ross River Virus.

UNLABELLED: The alphaviral6kgene region encodes the two structural proteins 6K protein and, due to a ribosomal frameshift event, the transframe protein (TF). Here, we characterized the role of the6kproteins in the arthritogenic alphavirus Ross River virus (RRV) in infected cells and in mice, using a novel6kin-frame deletion mutant. Comprehensive microscopic analysis revealed that the6kproteins were predominantly localized at the endoplasmic reticulum of RRV-infected cells. RRV virions that lack the6kproteins 6K and TF [RRV-(Δ6K)] were more vulnerable to changes in pH, and the corresponding virus had increased sensitivity to a higher temperature. While the6kdeletion did not reduce RRV particle production in BHK-21 cells, it affected virion release from the host cell. Subsequentin vivostudies demonstrated that RRV-(Δ6K) caused a milder disease than wild-type virus, with viral titers being reduced in infected mice. Immunization of mice with RRV-(Δ6K) resulted in a reduced viral load and accelerated viral elimination upon secondary infection with wild-type RRV or another alphavirus, chikungunya virus (CHIKV). Our results show that the6kproteins may contribute to alphaviral disease manifestations and suggest that manipulation of the6kgene may be a potential strategy to facilitate viral vaccine development. IMPORTANCE: Arthritogenic alphaviruses, such as chikungunya virus (CHIKV) and Ross River virus (RRV), cause epidemics of debilitating rheumatic disease in areas where they are endemic and can emerge in new regions worldwide. RRV is of considerable medical significance in Australia, where it is the leading cause of arboviral disease. The mechanisms by which alphaviruses persist and cause disease in the host are ill defined. This paper describes the phenotypic properties of an RRV6kdeletion mutant. The absence of the6kgene reduced virion release from infected cells and also reduced the severity of disease and viral titers in infected mice. Immunization with the mutant virus protected mice against viremia not only upon exposure to RRV but also upon challenge with CHIKV. These findings could lead to the development of safer and more immunogenic alphavirus vectors for vaccine delivery.

Pentosan Polysulfate: a Novel Glycosaminoglycan-Like Molecule for Effective Treatment of Alphavirus-Induced Cartilage Destruction and Inflammatory Disease.

UNLABELLED: Arthritogenic alphaviruses such as Ross River virus (RRV) and chikungunya virus (CHIKV) cause large-scale epidemics of severe musculoskeletal disease and have been progressively expanding their global distribution. Since its introduction in July 2014, CHIKV now circulates in the United States. The hallmark of alphavirus disease is crippling pain and inflammation of the joints, a similar immunopathology to rheumatoid arthritis. The use of glycans as novel therapeutics is an area of research that has increased in recent years. Here, we describe the promising therapeutic potential of the glycosaminoglycan (GAG)-like molecule pentosan polysulfate (PPS) to alleviate virus-induced arthritis. Mouse models of RRV and CHIKV disease were used to characterize the extent of cartilage damage in infection and investigate the potential of PPS to treat disease. This was assessed using histological analysis, real-time PCR, and fluorescence-activated cell sorting (FACS). Alphaviral infection resulted in cartilage destruction, the severity of which was alleviated by PPS therapy during RRV and CHIKV clinical disease. The reduction in cartilage damage corresponded with a significant reduction in immune infiltrates. Using multiplex bead arrays, PPS treatment was found to have significantly increased the anti-inflammatory cytokine interleukin-10 and reduced proinflammatory cytokines, typically correlated with disease severity. Furthermore, we reveal that the severe RRV-induced joint pathology, including thinning of articular cartilage and loss of proteoglycans in the cartilage matrix, was diminished with treatment. PPS is a promising new therapy for alphavirus-induced arthritis, acting to preserve the cartilage matrix, which is damaged during alphavirus infection. Overall, the data demonstrate the potential of glycotherapeutics as a new class of treatment for infectious arthritis. IMPORTANCE: The hallmark of alphavirus disease is crippling pain and joint arthritis, which often has an extended duration. In the past year, CHIKV has expanded into the Americas, with approximately 1 million cases reported to date, whereas RRV continues to circulate in the South Pacific. Currently, there is no licensed specific treatment for alphavirus disease, and the increasing spread of infection highlights an urgent need for therapeutic intervention strategies. Pentosan polysulfate (PPS) is a glycan derivative that is orally bioavailable, has few toxic side effects, and is currently licensed under the name Elmiron for the treatment of cystitis in the United States. Our findings show that RRV infection damages the articular cartilage, including a loss of proteoglycans within the joint. Furthermore, treatment with PPS reduced the severity of both RRV- and CHIKV-induced musculoskeletal disease, including a reduction in inflammation and joint swelling, suggesting that PPS is a promising candidate for drug repurposing for the treatment of alphavirus-induced arthritis.

Arthritogenic alphaviral infection perturbs osteoblast function and triggers pathologic bone loss.

Arthritogenic alphaviruses including Ross River virus (RRV), Sindbis virus, and chikungunya virus cause worldwide outbreaks of musculoskeletal disease. The ability of alphaviruses to induce bone pathologies remains poorly defined. Here we show that primary human osteoblasts (hOBs) can be productively infected by RRV. RRV-infected hOBs produced high levels of inflammatory cytokine including IL-6. The RANKL/OPG ratio was disrupted in the synovial fluid of RRV patients, and this was accompanied by an increase in serum Tartrate-resistant acid phosphatase 5b (TRAP5b) levels. Infection of bone cells with RRV was validated using an established RRV murine model. In wild-type mice, infectious virus was detected in the femur, tibia, patella, and foot, together with reduced bone volume in the tibial epiphysis and vertebrae detected by microcomputed tomographic (µCT) analysis. The RANKL/OPG ratio was also disrupted in mice infected with RRV; both this effect and the bone loss were blocked by treatment with an IL-6 neutralizing antibody. Collectively, these findings provide previously unidentified evidence that alphavirus infection induces bone loss and that OBs are capable of producing proinflammatory mediators during alphavirus-induced arthralgia. The perturbed RANKL/OPG ratio in RRV-infected OBs may therefore contribute to bone loss in alphavirus infection.

IL-3 and CSF-1 interact to promote generation of CD11c+ IL-10-producing macrophages.

Unraveling the mechanisms of hematopoiesis regulated by multiple cytokines remains a challenge in hematology. IL-3 is an allergic cytokine with the multilineage potential, while CSF-1 is produced in the steady state with restricted lineage coverage. Here, we uncovered an instructive role of CSF-1 in IL-3-mediated hematopoiesis. CSF-1 significantly promoted IL-3-driven CD11c+ cell expansion and dampened basophil and mast cell generation from C57BL/6 bone marrow. Further studies indicated that the CSF-1/CSF-1R axis contributed significantly to IL-3-induced CD11c+ cell generation through enhancing c-Fos-associated monopoiesis. CD11c+ cells induced by IL-3 or IL-3/CSF-1 were competent in cellular maturation and endocytosis. Both IL-3 and IL-3/CSF-1 cells lacked classical dendritic cell appearance and resembled macrophages in morphology. Both populations produced a high level of IL-10, in addition to IL-1, IL-6 and TNFα, in response to LPS, and were relatively poor T cell stimulators. Collectively, these findings reveal a role for CSF-1 in mediating the IL-3 hematopoietic pathway through monopoiesis, which regulates expansion of CD11c+ macrophages.

Enhanced Dendritic Cell-Mediated Antigen-Specific CD4+ T Cell Responses: IFN-Gamma Aids TLR Stimulation.

Phenotypic maturation and T cell stimulation are two functional attributes of DCs critical for immune induction. The combination of antigens, including those from cancer, with Toll-like receptor (TLR) ligands induces far superior cellular immune responses compared to antigen alone. In this study, IFN-gamma treatment of bone marrow-derived DC, followed by incubation with the TLR2, TLR4, or TLR9 agonists, enhanced DC activation compared to TLR ligation alone. Most notably, the upregulation of CD40 with LPS stimulation and CD86 with CpG stimulation was observed in in vitro cultures. Similarly, IFN-gamma coinjected with TLR ligands was able to promote DC activation in vivo, with DCs migrating from the site of immunization to the popliteal lymph nodes demonstrating increased expression of CD80 and CD86. The heightened DC activation translated to a drastic increase in T cell stimulatory capacity in both antigen independent and antigen dependent fashions. This is the first time that IFN-gamma has been shown to have a combined effect with TLR ligation to enhance DC activation and function. The results demonstrate the novel use of IFN-gamma together with TLR agonists to enhance antigen-specific T cell responses, for applications in the development of enhanced vaccines and drug targets against diseases including cancer.

A complementary role for the tetraspanins CD37 and Tssc6 in cellular immunity.

The cooperative nature of tetraspanin-tetraspanin interactions in membrane organization suggests functional overlap is likely to be important in tetraspanin biology. Previous functional studies of the tetraspanins CD37 and Tssc6 in the immune system found that both CD37 and Tssc6 regulate T cell proliferative responses in vitro. CD37(-/-) mice also displayed a hyper-stimulatory dendritic cell phenotype and dysregulated humoral responses. In this study, we characterize "double knockout" mice (CD37(-/-)Tssc6(-/-)) generated to investigate functional overlap between these tetraspanins. Strong evidence for a cooperative role for these two proteins was identified in cellular immunity, where both in vitro T cell proliferative responses and dendritic cell stimulation capacity are significantly exaggerated in CD37(-/-)Tssc6(-/-) mice when compared with single knockout counterparts. Despite these exaggerated cellular responses in vitro, CD37(-/-)Tssc6(-/-) mice are not more susceptible to autoimmune induction. However, in vivo responses to pathogens appear poor in CD37(-/-)Tssc6(-/-) mice, which showed a reduced ability to produce influenza-specific T cells and displayed a rapid onset hyper-parasitemia when infected with Plasmodium yoelii. Therefore, in the absence of both CD37 and Tssc6, immune function is further altered when compared with CD37(-/-) or Tssc6(-/-) mice, demonstrating a complementary role for these two molecules in cellular immunity.

Methods to measure T-cell responses.

A successful vaccine for immunotherapy, particularly for solid tumors or viral infections, requires a suitable target antigen and the production of a cytotoxic T-cell response. In addition, CD4 T cells play an important role in cellular immunity. Here, we briefly discuss methods by which T cells are measured in vitro after vaccination.

Reactive oxygen species level defines two functionally distinctive stages of inflammatory dendritic cell development from mouse bone marrow.

Reactive oxygen species (ROS) have been implicated in various physiological activities. However, their role in dendritic cell (DC) activation and generation has not been investigated. Using the bone marrow-derived GM-CSF-induced ex vivo DC model, we characterize how induction of ROS correlates with inflammatory DC functionality and expansion. We describe that the functionality of GM-CSF-induced DCs is distinct in two developmental stages. Whereas division of DC-committed hematopoietic progenitor cells (HPCs) neared completion by day 6, the level of ROS soared after day 4. Day 3 ROS(lo) DCs were highly responsive to TLR stimuli such as LPS and zymosan by rapid upregulation of CD80, CD86, and MHC class II, in contrast to the low response of day 6 ROS(hi) DCs. ROS(hi) DCs could not initiate and sustain a significant level of NF-kappaB phosphorylation in response to LPS and zymosan, although demonstrating hyperactivation of p38 MAPK by LPS, in a fashion disparate to ROS(lo) DCs. ROS(lo) DCs stimulated a higher level of allogeneic and OVA-specific T cell proliferative responses, although ROS(hi) DCs were much more proficient in processing OVA. In response to pathogenic stimuli, ROS(hi) DCs also demonstrated rapid cellular adhesion and H(2)O(2) release, indicating their role in immediate microbial targeting. Moreover, HPC expansion and DC generation were dependent on the surge of ROS in an NADPH oxidase-independent manner. These findings point to the potential role of cellular ROS in mediating functionality and development of DCs from HPCs during inflammation.

Tetraspanins CD37 and CD151 differentially regulate Ag presentation and T-cell co-stimulation by DC.

A major question in immunology is how DC can display limited amounts of individual peptide-MHC complexes and still induce cross-linking of T-cell receptors to initiate cellular responses. One suggested mechanism is that MHC exists at the cell surface in high avidity multimers, and tetraspanin proteins, known to laterally associate with both MHC classes I and II, promote MHC multimerisation. To validate this theory, we tested the ability of DC deficient in either one of two typical tetraspanin molecules: CD37 or CD151 to present peptide to Ag-specific T cells. Surprisingly, although they exhibited no developmental or maturation defects, DC lacking either CD37 or CD151 expression were hyper-stimulatory to T cells. We demonstrate that CD37 and CD151 control DC-mediated T-cell activation by two different mechanisms: CD151 regulates co-stimulation whereas CD37 regulates peptide/MHC presentation. The implications of these results on the model suggesting that tetraspanins promote MHC multimerisation are discussed.

The adjuvanticity of a mannosylated antigen reveals TLR4 functionality essential for subset specialization and functional maturation of mouse dendritic cells.

The evidence that dendritic cell (DC) subsets produce differential cytokines in response to specific TLR stimulation is robust. However, the role of TLR stimulation in Ag presentation and phenotypic maturation among DC subsets is not clear. Through the adjuvanticity of a novel mannosylated Ag, mannosylated dendrimer OVA (MDO), as a pathogen-associated molecular pattern Ag, we characterized the functionality of GM-CSF/IL-4-cultured bone marrow DC and Flt3 ligand (Flt3-L) DC subsets by Ag presentation and maturation assays. It was demonstrated that both bone marrow DCs and Flt3-L DCs bound, processed, and presented MDO effectively. However, while Flt3-L CD24(high) (conventional CD8(+) equivalent) and CD11b(high) (CD8(-) equivalent) DCs were adept at MDO processing by MHC class I and II pathways, respectively, CD45RA(+) plasmacytoid DCs presented MDO poorly to T cells. Successful MDO presentation was largely dependent on competent TLR4 for Ag localization and morphological/phenotypic maturation of DC subsets, despite the indirect interaction of MDO with TLR4. Furthermore, Toll/IL-1 receptor-domain-containing adaptor-inducing IFN-beta, but not MyD88, as a TLR4 signaling modulator was indispensable for MDO-induced DC maturation and Ag presentation. Taken together, our findings suggest that DC subsets differentially respond to a pathogen-associated molecular pattern-associated Ag depending on the intrinsic programming and TLRs expressed. Optimal functionality of DC subsets in Ag presentation necessitates concomitant TLR signaling critical for efficient Ag localization and processing.

Oxidized and reduced mannan mediated MUC1 DNA immunization induce effective anti-tumor responses.

DNA immunization is an attractive form of vaccination, which has shown promising results only in small animal models. There is a need to develop efficient gene delivery systems. We previously demonstrated that oxidized (OM) and reduced mannan (RM) complexed to ovalbumin DNA via poly-l-lysine (PLL), were able to generate potent immune responses in mice. Herein, we further investigated the suitability of OMPLL and RMPLL as carriers for mucin 1 (MUC1) DNA vaccination for cancer immunotherapy. Studies presented here showed that immune responses in C57BL/6 mice induced by OMPLL-MUC1 DNA and RMPLL-MUC1 DNA immunization were more immunogenic compared to MUC1 DNA alone. Moreover, tumor protection was evident at a dose as low as 0.5 microg. In addition, strong T cell responses were induced in HLA-A2 transgenic and human MUC1 transgenic mice. These results demonstrate the potential of OM and RM as efficient non-viral gene delivery carriers for DNA vaccines for use in cancer immunotherapy.

Delivery of antigen using a novel mannosylated dendrimer potentiates immunogenicity in vitro and in vivo.

Antigen mannosylation has been shown to be an effective approach to potentiate antigen immunogenicity, due to the enhanced antigen uptake and presentation by APC. To overcome disadvantages associated with conventional methods used to mannosylate antigens, we have developed a novel mannose-based antigen delivery system that utilizes a polyamidoamine (PAMAM) dendrimer. It is demonstrated that mannosylated dendrimer ovalbumin (MDO) is a potent immune inducer. With a strong binding avidity to DC, MDO potently induced OVA-specific T cell response in vitro. It was found that the immunogenicity of MDO was due not only to enhanced antigen presentation, but also to induction of DC maturation. Mice immunized with MDO generated strong OVA-specific CD4(+)/CD8(+) T cell and antibody responses. MDO also targeted lymph node DC to cross-present OVA, leading to OTI CD8(+) T cell proliferation. Moreover, upon challenge with B16-OVA tumor cells, tumors in mice pre-immunized with MDO either did not grow or displayed a much more delayed onset, and had slower kinetics of growth than those of OVA-immunized mice. This mannose-based antigen delivery system was applied here for the first time to the immunization study. With several advantages and exceptional adjuvanticity, we propose mannosylated dendrimer as a potential vaccine carrier.

Dendritic cell vaccination.

There has been a surge of interest in the use of dendritic cell (DC) vaccination as cellular immunotherapy for numerous cancers. Despite some encouraging results, this therapeutic modality is far from being considered as a therapy for cancer. This review will first discuss preclinical DC vaccination in murine models of cancer, with an emphasis on comparative studies investigating different methods of antigen priming. We will then comment on the various murine DC subsets and how these relate to human DC preparations used for clinical studies. Finally, the methodology used to generate human DCs and some recent clinical trials in several cancers are reviewed.

Mannan derivatives induce phenotypic and functional maturation of mouse dendritic cells.

Mannan, a polysaccharide isolated from yeast binds to C-type lectins of the mannose receptor family, expressed by antigen-presenting cells (APCs) including dendritic cells (DCs) and macrophages. As these receptors mediate endocytosis, they have been targeted with ligands to deliver antigens into APCs to initiate immune responses. Immunization with tumour antigen MUC1 conjugated to oxidized mannan (OM) or reduced mannan (RM) induced differential immune responses in mice, and only mice immunized with OM-MUC1 elicited strong MUC1-specific cytotoxic T lymphocyte responses and protected mice from a MUC1 tumour challenge. In this study, the adjuvant effect of mannan and its derivatives including OM and RM, in comparison to lipopolysaccharide, on DCs were investigated. Mannan, OM and RM were capable of stimulating mouse bone marrow-derived DC in vitro, eliciting enhanced allogeneic T-cell proliferation and enhancing OTI/OTII peptide-specific T-cell responses. Injection of mice with mannan, OM and RM induced a mature phenotype of lymph node and splenic DCs. Analysis by reverse transcription-polymerase chain reaction indicated that Manna, OM and RM also stimulated up-regulation of inflammatory cytokines including interleukin-1beta and tumour necrosis factor-alpha, and differential T helper 1 (Th1)/Th2 cytokines. Subsequent experiments demonstrated that activation of DCs was Toll-like receptor-4-dependent. The data presented here, together with evidence reported previously on OM and RM in induction of immune responses in vivo, suggest that OM and RM exert a dual capacity to target antigen to APCs as well as mature DCs.

Dendritic cells: activation and maturation--applications for cancer immunotherapy.

There is an increasing number of studies utilizing dendritic cell (DC) based therapies for cancer. With a powerful antigen-presentation capability, DCs have the potential to overcome tumor tolerance and induce anti-tumor immunity, when loaded with tumor antigens. In order to optimize this approach, methods have aimed to enhance immunopotency of therapeutic DCs. A thorough understanding of DC immunobiology would accelerate this process and provide advantageous procedures to increase anti-tumor responses. This review contains an analysis of recent advances on DC subsets, phenotypic characterization, localization, surface receptors and their ligands. The events of immune induction via DCs, involving initial recognition and uptake of antigens, migration, subsequent activation and maturation are revisited. Furthermore, the current methods used for DC-based cancer immunotherapy, including DCs pulsed with tumor antigens in forms of DNA, RNA, peptides, proteins and lysates, or DCs fused with tumor cells are summarized. Respective preclinical and clinical trials are in progress and hold promise for developing effective cancer vaccines.

Characterization of mice lacking the tetraspanin superfamily member CD151.

The tetraspanin membrane protein CD151 is a broadly expressed molecule noted for its strong molecular associations with integrins, especially alpha3beta1, alpha6beta1, alpha7beta1, and alpha6beta4. In vitro functional studies have pointed to a role for CD151 in cell-cell adhesion, cell migration, platelet aggregation, and angiogenesis. It has also been implicated in epithelial tumor progression and metastasis. Here we describe the generation and initial characterization of CD151-null mice. The mice are viable, healthy, and fertile and show normal Mendelian inheritance. They have essentially normal blood and bone marrow cell counts and grossly normal tissue morphology, including hemidesmosomes in skin, and expression of alpha3 and alpha6 integrins. However, the CD151-null mice do show phenotypes in several different tissue types. An absence of CD151 leads to a minor abnormality in hemostasis, with CD151-null mice showing longer average bleeding times, greater average blood loss, and an increased incidence of rebleeding occurrences. CD151-null keratinocytes migrate poorly in skin explant cultures. Finally, CD151-null T lymphocytes are hyperproliferative in response to in vitro mitogenic stimulation.