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Objective Divergent therapeutic outcomes on different disease domains have been noted with IL-23 and IL-17A-blockade in PsA. Therefore, elucidating the role of RORγt, the master regulator of type 17 immune responses, is of potential therapeutic interest. To this end, RORγt inhibition was assessed in combined skin, joint and gut inflammation in vivo, using a PsA model. Methods We tested the efficacy of a RORγt antagonist in B10.RIII mice challenged with systemic overexpression of IL-23 by hydrodynamic injection of IL-23 enhanced episomal vector (IL-23 EEV). Clinical outcomes were evaluated by histopathology. Bone density and surface erosions were examined using micro-computed tomography. Cytokine production was measured in serum and by intracellular flow cytometry. Gene expression in PsA-related tissues was analysed by qPCR. Results RORγt-blockade significantly ameliorated psoriasis, peripheral arthritis and colitis development in IL-23 EEV mice (improvement of clinical scores and weight loss respectively by 91.8%, 58.2% and 7.0%, P Conclusion RORγt-blockade shows therapeutic efficacy in a preclinical PsA model with protection towards extra-musculoskeletal manifestations, reflected by a clear attenuation of type 17 cytokine responses by γδ-T cells and Th17 cells.

The anti-inflammatory protein A20 serves as a critical brake on NF-kappa B signaling and NF-kappa B-dependent inflammation. In humans, polymorphisms in or near the TNFAIP3/A20 gene have been associated with several inflammatory disorders, including rheumatoid arthritis (RA), and experimental studies in mice have demonstrated that myeloid-specific A20 deficiency causes the development of a severe polyarthritis resembling human RA. Myeloid A20 deficiency also promotes osteoclastogenesis in mice, suggesting a role for A20 in the regulation of osteoclast differentiation and bone formation. We show here that osteoclast-specific A20 knockout mice develop severe osteoporosis, but not inflammatory arthritis. In vitro, osteoclast precursor cells from A20 deficient mice are hyper-responsive to RANKL-induced osteoclastogenesis. Mechanistically, we show that A20 is recruited to the RANK receptor complex within minutes of ligand binding, where it restrains NF-kappa B activation independently of its deubiquitinating activity but through its zinc finger (ZnF) 4 and 7 ubiquitin-binding functions. Together, these data demonstrate that A20 acts as a regulator of RANK-induced NF-kappa B signaling to control osteoclast differentiation, assuring proper bone development and turnover.

Objective: Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT-1) plays a crucial role in innate and adaptive immune signaling by modulating the threshold for activation of immune cells, including Treg cells. Therefore, MALT-1 is regarded to be an interesting therapeutic target in several immune-mediated diseases. The goal of this study was to examine the role of MALT-1 in experimental animal models of rheumatoid arthritis (RA). Methods: MALT-1 activation was assessed by measuring cleavage of the deubiquitinase CYLD in lymphocytes from mice with collagen-induced arthritis (CIA). Furthermore, the impact of MALT-1 deficiency on arthritis was evaluated in Malt1(KO) mice with CIA or with collagen antibody-induced arthritis (CAIA). T cell-specific MALT-1 deficiency was measured in mice with deletion of T cell-specific MALT-1 (Malt1(TcellKO) ), and the time-dependent effects of MALT-1 deficiency were assessed in mice with deletion of tamoxifen-inducible T cell-specific MALT-1 (Malt1(iTcellKO)). Bone density was determined in MALT-1-deficient mice using micro-computed tomography and femur-bending tests. Reconstitution of Treg cells was performed using adoptive transfer experiments. Results: MALT-1 activation was observed in the lymphocytes of mice with CIA. T cell-specific MALT-1 deletion in the induction phase of arthritis (incidence of arthritis, 25% in control mice versus 0% in Malt1(iTcellKO) mice; P < 0.05), but not in the effector phase of arthritis, completely protected mice against the development of CIA. Consistent with this finding, MALT-1 deficiency had no impact on CAIA, an effector phase model of RA. Finally, mice with MALT-1 deficiency showed a spontaneous decrease in bone density (mean SEM trabecular thickness, 46.3 +/- 0.7 mu m in control mice versus 40 +/- 1.1 mu m in Malt1(KO) mice; P < 0.001), which was linked to the loss of Treg cells in these mice. Conclusion: Overall, these data in murine models of RA highlight MALT-1 as a master regulator of T cell activation, which is relevant to the pathogenesis of autoimmune arthritis. Furthermore, these findings show that MALT-1 deficiency can lead to spontaneous osteoporosis, which is associated with impaired Treg cell numbers.

Background Alterations in ambient temperature have been associated with multiple detrimental effects on broilers such as intestinal barrier disruption and dysbiosis resulting in systemic inflammation. Inflammation and 25-hydroxycholecalciferol (25-OH-D3) have shown to play a negative and positive role, respectively, in the regulation of bone mass. Hence the potential of 25-OH-D3 in alleviating heat induced bone alterations and its mechanisms was studied. Results Heat stress (HS) directly induced a decrease in tibia material properties and bone mass, as demonstrated by lower mineral content, and HS caused a notable increase in intestinal permeability. Treatment with dietary 25-OH-D3 reversed the HS-induced bone loss and barrier leak. Broilers suffering from HS exhibited dysbiosis and increased expression of inflammatory cytokines in the ileum and bone marrow, as well as increased osteoclast number and activity. The changes were prevented by dietary 25-OH-D3 administration. Specifically, dietary 25-OH-D3 addition decreased abundance of B- and T-cells in blood, and the expression of inflammatory cytokines, especially TNF-α, in both the ileum and bone marrow, but did not alter the diversity and population or composition of major bacterial phyla. With regard to bone remodeling, dietary 25-OH-D3 supplementation was linked to a decrease in serum C-terminal cross-linked telopeptide of type I collagen reflecting bone resorption and a concomitant decrement in osteoclast-specific marker genes expression (e.g. cathepsin K), whereas it did not apparently change serum bone formation markers during HS. Conclusions These data underscore the damage of HS to intestinal integrity and bone health, as well as that dietary 25-OH-D3 supplementation was identified as a potential therapy for preventing these adverse effects.

CD1d‐restricted invariant natural killer T (iNKT) cells constitute a common glycolipid‐reactive innate‐like T‐cell subset with a broad impact on innate and adaptive immunity. While several microbial glycolipids are known to activate iNKT cells, the cellular mechanisms leading to endogenous CD1d‐dependent glycolipid responses remain largely unclear. Here, we show that endoplasmic reticulum (ER) stress in APCs is a potent inducer of CD1d‐dependent iNKT cell autoreactivity. This pathway relies on the presence of two transducers of the unfolded protein response: inositol‐requiring enzyme‐1a (IRE1α) and protein kinase R‐like ER kinase (PERK). Surprisingly, the neutral but not the polar lipids generated within APCs undergoing ER stress are capable of activating iNKT cells. These data reveal that ER stress is an important mechanism to elicit endogenous CD1d‐restricted iNKT cell responses through induction of distinct classes of neutral lipids., Upon ER stress immunogenic lipids are produced that activate NKT cells. Defining the conditions required to generate endogenous high immunogenic lipid antigens paves the way for the design of novel lipids with immunomodulatory function.

ObjectivesThe mechanisms driving onset of joint inflammation in arthritides such as rheumatoid arthritis and spondyloarthritis and the conversion to disease chronicity are poorly understood. We hypothesised mechanostrain could play an instrumental role herein by engaging local and/or systemic pathways, thereby attenuating disease course and outcome.MethodsThe development of collagen antibody-induced arthritis (CAIA) in C57BL/6 mice was evaluated both clinically and histologically under different loading regimens: control, voluntary running or hindpaw unloading. Bone surface porosity was quantified by high-resolution µ-CT. Gene expression analyses were conducted by microarrays and qPCR on microdissected entheses, murine and human synovial tissues (both normal and inflamed). Serum cytokines and chemokines were measured by ELISA. The influence of complement activation and T regulatory (Treg) cell function on the induction and resolution phase of disease was studied by respectively pharmacological modulation and conditional Treg depletion.ResultsVoluntary running strongly impacts the course of arthritis by impairing the resolution phase of CAIA, leading to more persistent inflammation and bone surface porosity. Mechanical strain induced local complement activation, increased danger-associated molecular pattern expression, activating Fcγ receptors as well as changes in fibroblast phenotype. Interestingly, complement C5a receptor blockade inhibited the enhanced joint pathology caused by voluntary running. Moreover, Treg depletion led to a loss of disease resolution in CAIA mice, which was not observed under voluntary running conditions.ConclusionsRunning promotes onset and chronicity of arthritis by local upregulation of complement activators and hampering regulatory T cell feedback loops.

Multiple myeloma (MM) is a progressive monoclonal B cell malignancy, for which survival and progression largely relies on the crosstalk of tumor cells with the bone marrow (BM) microenvironment, inducing immune escape, angiogenesis, bone destruction and drug resistance. Despite great therapeutic advances, most of the MM patients still relapse and remain incurable. Over the past years, immunotherapy has emerged as a new field in cancer therapy. Here, the immune cells of the patients themselves are activated to target the tumor cells. In MM, several effector cells of the immune system are present in the BM microenvironment; unfortunately, they are mostly all functionally impaired. In this review, we focus on the role of innate-like T cells in MM, particularly CD1d- and MR1- restricted T cells such as respectively invariant natural killer T (iNKT) cells and mucosal associated invariant T (MAIT) cells. These cells have the capacity upon activation to rapidly release copious amounts of cytokines affecting a wide range of innate and adaptive immune responses, and could therefore play a key protective role in anti-tumor immunity. We describe recent observations with regard to functional exhaustion of iNKT and MAIT cells in MM pathology and discuss the potential application of checkpoint inhibition as an attractive target for prolonged activation of these immunomodulatory T cells in the treatment of MM.

Background Many pro-inflammatory pathways leading to arthritis act systemically on the immune system rather than locally in the joint. However, the reason behind the regional and patchy distribution of arthritis represents a longstanding paradox. Objectives To explore the relation between mechanical strain and joint inflammation and to understand the underlying basis of joint pattern involvement in inflammatory rheumatic diseases. Methods Arthritis was induced by collagen-induced arthritis (CIA) and passive collagen antibody induced arthritis (CAIA) in respectively C57BL/6 and RAG2-/- (T- and B-cell deficient) mice. Animals were subjected to different regimens of mechanical strain. Increased strain occurred in voluntary running mice whereas tail suspension (unloading) abolished mechanical strain; both were compared to control housing conditions. The impact of different loading conditions was measured on clinical disease score, histology, micro-CT images and erosion quantification, gene induction in tendon and synovial tissue, immune cell recruitment in situ, development of anti-collagen antibodies and their pattern of siaylation and galactosylation. Results Voluntary running of CIA in C57BL/6 mice markedly induced an early onset and increased progression whereas no disease onset could be observed in the hind paws from animals in unloaded conditions. CAIA in running RAG2-/- mice also induced early arthritic symptoms and severe progression. Intriguingly, running conditions were sufficient to induce arthritis without the need of LPS as an inflammatory trigger. Mechanical strain did not alter however IgG autoantibody levels nor their levels of galactosylation and sialylation. Furthermore, we demonstrate that mechanical strain on stromal cells results in recruitment of classical monocytes into specialised mechano-sensitive regions characterised by a unique microanatomy. This promotes local inflammation and differentiation into local osteoclasts which induce regional erosions. A striking similarity was observed in the pattern of joint erosions in human patients with RA and SpA which were also confined to these mechanosensitive regions. Conclusions This study provides the first evidence that mechanical strain controls the transition from systemic autoimmunity into site-specific joint inflammation. Homing of inflammation and development of erosions was confined to specific mechano-sensitive regions, characterised by a high number of attachment- and contact points for tendons. This represents a novel paradigm and explains why arthritis in mice and humans is characterised by a regional and patchy distribution. Curiously, this pathway does not rely on adaptive immunity but rather on stromal cells. Mechano-stimulation of mesenchymal cells induced CXCL1 and CCL2 permitting recruitment of classical monocytes which can differentiate into bone-resorbing osteoclasts. Thus, mechanical strain controls the site-specific direction of inflammation and tissue damage in arthritis. Acknowledgements Disclosure of Interest None declared

Background Oxygen supply is a fundamental requirement for all living tissues. Some tissues such as articular joints are characterized by a physiological state of hypoxia. Interestingly, under conditions of inflammation such as in arthritic disease, this level of hypoxia is even further enhanced. However, the functional significance of these observations and the molecular mechanisms involved remain poorly characterized to date. Our goal was therefore to examine the role of 3 known oxygen sensors, prolyl hydroxylase domain (PHD) proteins: PHD1, PHD2 and PHD3. They are enzymes whose function is essentially controlled by oxygen. Their expression pattern varies between either of them and all of them have been ascribed specific roles in a myriad of biological processes. [1] Objectives Our goal was to examine the role of oxygen sensors PHD1, PHD2 and PHD3 in preclinical models of rheumatoid arthritis, and to delineate the cellular source involved. Methods We subjected the collagen antibody induced arthritis (CAIA) model (resembling rheumatoid arthritis) to hypoxic (10% O2) and normoxic conditions (21% O2), respectively. Furthermore, the CAIA-model was induced in mice with germline deficiency of the specific PHD9s and in mice with a myeloid cell-specific PHD1 deficiency versus controls. Arthritis development was assessed by clinical scoring of paw swelling, histopathology of knee joints and μCT. Results Mice kept in hypoxic conditions during CAIA experiments showed markedly less arthritis (both by clinical and histopathological assessment) compared to mice in normoxic conditions. Furthermore, we demonstrated that PHD1 knock-out (KO) mice had significantly less joint inflammation compared to wildtype mice. PHD1 KO mice were also protected against inflammation induced bone loss as evidenced by μCT. By contrast, no differences were found between PHD2 heterozygous (PHD2 KO mice are not viable) or PHD3 KO mice and littermate controls. Because myeloid cells are considered critical effector cells upon passive transfer of arthritogenic antibodies in the CAIA model we also generated myeloid cell specific ko mice (PHD1myelKO). Of interest, PHD1myelKO mice developed less arthritis compared to wildtype mice and were protected against inflammation induced bone loss. Conclusions Our data are consistent with a new paradigm that the oxygen sensor PHD1 is a critical regulator of myeloid cell function in arthritic disease. Overall, the data suggest that PHD1 is a potential target in the treatment of arthritis. References Fong G-H, Takeda K. Role and regulation of prolyl hydroxylase domain proteins. Cell Death Differ 2008;15:635–41. doi:10.1038/cdd.2008.10. Disclosure of Interest None declared

A hallmark of bone marrow changes with aging is the increase in adipocyte composition, but how this impacts development of multiple myeloma (MM) is unknown. Here, we report the role of the adipokine leptin as master regulator of anti-myeloma tumor immunity by modulating the invariant natural killer T (iNKT) cell function. A marked increase in serum leptin levels and leptin receptor (LR) expression on iNKT cells in MM patients and the 5T33 murine MM model was observed. MM cells and leptin synergistically counteracted anti-tumor functionality of both murine and human iNKT cells. In vivo blockade of LR signaling combined with iNKT stimulation resulted in superior anti-tumor protection. This was linked to persistent IFN-γ secretion upon repeated iNKT cell stimulation and a restoration of the dynamic antigen-induced motility arrest as observed by intravital microscopy, thereby showing alleviation of iNKT cell anergy. Overall our data reveal the LR axis as novel therapeutic target for checkpoint inhibition to treatMM.Leukemia advance online publication, 16 June 2017; doi:10.1038/leu.2017.146.

Summary Macrophages are strongly adapted to their tissue of residence. Yet, little is known about the cell-cell interactions that imprint the tissue-specific identities of macrophages in their respective niches. Using conditional depletion of liver Kupffer cells, we traced the developmental stages of monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting the Kupffer cell identity. Kupffer cell loss induced tumor necrosis factor (TNF)- and interleukin-1 (IL-1) receptor-dependent activation of stellate cells and endothelial cells, resulting in the transient production of chemokines and adhesion molecules orchestrating monocyte engraftment. Engrafted circulating monocytes transmigrated into the perisinusoidal space and acquired the liver-associated transcription factors inhibitor of DNA 3 (ID3) and liver X receptor-α (LXR-α). Coordinated interactions with hepatocytes induced ID3 expression, whereas endothelial cells and stellate cells induced LXR-α via a synergistic NOTCH-BMP pathway. This study shows that the Kupffer cell niche is composed of stellate cells, hepatocytes, and endothelial cells that together imprint the liver-specific macrophage identity., Graphical Abstract, Highlights • Kupffer cells interact with stellate cells and hepatocytes in the space of Disse • TNF released by dying Kupffer cells activates stellate cells and endothelial cells • Stellate cells and endothelial cells orchestrate monocyte recruitment and adhesion • Endothelial and stellate cells induce LXR-α in monocytes via the NOTCH-BMP pathway, Bonnardel and colleagues tracked monocytes differentiating into Kupffer cells and mapped the cellular interactions imprinting their identity. Monocytes transmigrated into the perisinusoidal space. Interactions with endothelial and stellate cells induced LXR-α via the NOTCH-BMP pathway. Hepatocytes induced ID3. Thus, stellate cells, hepatocytes, and endothelial cells compose the Kupffer cell niche.

Glaucomatous optic neuropathies are characterized by progressive loss of retinal ganglion cells (RGCs), the neurons that connect the eye to the brain. Quantification of these RGCs is a cornerstone in experimental optic neuropathy research and commonly performed via manually quantifying parts of the retina. However, this is a time-consuming process subject to inter- and intra-observer variability. Here we present a freely available ImageJ script to semi-automatically quantify RGCs in entire retinal flatmounts after immunostaining for the RGC-specific transcription factor Brn3a. The blob-like signal of Brn3a-immunopositive RGCs is enhanced via eigenvalues of the Hessian matrix and the resulting local maxima are counted as RGCs. After the user has outlined the retinal flatmount area, the total RGC number and retinal area are reported and an isodensity map, showing the RGC density distribution across the retina, is created. The semi-automated quantification shows a very strong correlation (Pearson's r ≥ 0.99) with manual counts for both widefield and confocal images, thereby validating the data generated via the developed script. Moreover, application of this method in established glaucomatous optic neuropathy models such as N-methyl-D-aspartate-induced excitotoxicity, optic nerve crush and laser-induced ocular hypertension revealed RGC loss conform with literature. Compared to manual counting, the described automated quantification method is faster and shows user-independent consistency. Furthermore, as the script detects the RGC number in entire retinal flatmounts, the method allows detection of regional differences in RGC density. As such, it can help advance research investigating the degenerative mechanisms of glaucomatous optic neuropathies and the effectiveness of new neuroprotective treatments. Because the script is flexible and easy to optimize due to a low number of critical parameters, it can potentially be applied in combination with other tissues or alternative labeling protocols.

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system (CNS) remains elusive. The discovery of novel neuro-stimulatory agents that promote regeneration is hampered by a gap between high content analysis platforms using neuronal cells and time-consuming preclinical animal models. In this regard, tissue explant cultures, which are easily manageable and more closely resemble the in vivo situation, form an ideal model system to study the effect of compounds on the neuroglial network. Retinal explants have proven to be a useful tool to investigate the effect of molecules on neuronal survival and regeneration. In this chapter, we report a detailed description of how to isolate and culture retinal explants and how to immunolabel the outgrowing neurites. Furthermore, we describe different analysis tools, both manual and automated, to quantify neurite outgrowth from retinal explants.

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system (CNS) remains elusive. The discovery of novel neuro-stimulatory agents that promote regeneration is hampered by a gap between high content analysis platforms using neuronal cells and time-consuming preclinical animal models. In this regard, tissue explant cultures, which are easily manageable and more closely resemble the in vivo situation, form an ideal model system to study the effect of compounds on the neuroglial network. Retinal explants have proven to be a useful tool to investigate the effect of molecules on neuronal survival and regeneration. In this chapter, we report a detailed description of how to isolate and culture retinal explants and how to immunolabel the outgrowing neurites. Furthermore, we describe different analysis tools, both manual and automated, to quantify neurite outgrowth from retinal explants. ispartof: Axon Growth and Regeneration: Methods and Protocols pages:57-71 ispartof: Axon Growth and Regeneration: Methods and Protocols (Methods in Molecular Biology Series) vol:1162 pages:57-71 ispartof: location:United States status: published

Restoration of correct neural activity following central nervous system (CNS) damage requires the replacement of degenerated axons with newly outgrowing, functional axons. Unfortunately, spontaneous regeneration is largely lacking in the adult mammalian CNS. In order to establish successful regenerative therapies, an improved understanding of axonal outgrowth and the various molecules influencing it, is highly needed. Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases that were sporadically reported to influence axon outgrowth. Using an ex vivo retinal explant model, we were able to show that broad-spectrum MMP inhibition reduces axon outgrowth of mouse retinal ganglion cells (RGCs), implicating MMPs as beneficial factors in axonal regeneration. Additional studies, using more specific MMP inhibitors and MMP-deficient mice, disclosed that both MMP-2 and MT1-MMP, but not MMP-9, are involved in this process. Furthermore, administration of a novel antibody to MT1-MMP that selectively blocks pro-MMP-2 activation revealed a functional co-involvement of these proteinases in determining RGC axon outgrowth. Subsequent immunostainings showed expression of both MMP-2 and MT1-MMP in RGC axons and glial cells. Finally, results from combined inhibition of MMP-2 and β1-integrin were suggestive for a functional interaction between these molecules. Overall, our data indicate MMP-2 and MT1-MMP as promising axonal outgrowth-promoting molecules. Axonal regeneration in the central nervous system is lacking in adult mammals, thereby impeding recovery from injury to the nervous system. Matrix metalloproteinases (MMPs) constitute a family of zinc-dependent proteases that were sporadically reported to influence axon outgrowth. Inhibition of specific MMPs reduced neurite outgrowth from mouse retinal explants. Our data indicate MMP-2 and MT1-MMP as promising axonal outgrowth-promoting molecules and show a possible link between MMP-2 and β1-integrin in axon outgrowth.

Despite intensive research efforts over the past years, regeneration of injured axons in the central nervous system remains elusive. In the quest for neurostimulatory agents that promote regeneration, well-defined models and analysis methods are required. Tissue explant cultures closely resemble the in vivo situation, making them ideal to study the effect of compounds on the neuro-glial network. This study reports the optimization of an explant culture technique using retinas of neonatal mice and the development of an analysis script that allows for rapid and automated analysis of neurite outgrowth from these explants. The key features of this script (i.e., local thresholding and form selection) allow for swift and unbiased detection of neurite outgrowth. The novel analysis method is compared with two commonly used manual methods and successfully validated by performing dose-response studies with molecules known to either inhibit (anti-β1-integrin antibody) or stimulate (brain-derived neurotrophic factor and ciliary neurotrophic factor) neurite outgrowth from retinal explants. Finally, the new analysis script is used to study whether retinal explant origin has any effect on neurite outgrowth.

Purpose Multiple studies in glaucoma patients and animal models, have reported differential expression and activity of matrix metalloproteinases (MMPs). These data have led to the hypothesis that MMPs are involved in glaucoma disease onset and/or progression. However, their in vivo functions remain poorly understood and contradictorily results prevent a clear definition of their role. Here, we describe the expression of MMP-2, -3, -9 and -14 in the retina of mice subjected to an acute excitotoxic glaucoma model. Methods Excitotoxic RGC death was induced via intravitreal injection of 20 mM NMDA. Expression of MMP-2, -3, -9 and -14 was examined via immunohistochemistry, Western blot and quantitative RT-PCR. Results MMP-2 and -3 are expressed by glia, presumably Muller glia, in the healthy retina and are strongly upregulated at 24h after NMDA injection. MMP-9 expression, which is not detectable in naive retinas, is observed in RGCs at 24h post NMDA injection, which confirms its suggested role in RGC apoptosis. In the naive retina, MMP-14 is expressed by bundles of RGC axons in the nerve fiber layer, from where its expression is extending through the optic nerve to the primary visual areas in the brain. Within the first 48h after NMDA injection, MMP-14 expression increases and is also seen in the inner nuclear layer and both plexiform layers. Conclusion Our results reveal a strong upregulation of MMP-2, -3, -9 and -14 in the mouse retina after NMDA injection, suggesting that these proteinases might be involved in excitotoxic neurodegeneration and/or glial reactivity. Further analysis of their involvement, including studies in MMP knockout mice, is currently ongoing.