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Fibroblast-like synoviocytes (FLS) play an important pathological role in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). These cells have primarily been characterized in the RA synovial membrane. Here we aim to phenotypically and functionally characterize cultured synovial fluid-derived FLS (sfRA-FLS). Paired peripheral blood mononuclear cells (PBMC) and sfRA-FLS from patients with RA were obtained and monocultures of sfRA-FLS and autologous co-cultures of sfRA-FLS and PBMC were established. The in situ activated sfRA-FLS were CD34-, CD45-, Podoplanin+, Thymocyte differentiation antigen-1+. SfRA-FLS expressed uniform levels of NFкB-related pathway proteins and secreted several pro-inflammatory cytokines dominated by IL-6 and MCP-1. In a co-culture model with autologous PBMC, the ICAM-1 and HLA-DR expression on sfRA-FLS and secretion of IL-1β, IL-6, and MCP-1 increased. In vivo, human sfRA-FLS were cartilage invasive both at ipsilateral and contralateral implantation site. We conclude that, sfRA-FLS closely resemble the pathological sublining layer FLS subset in terms of surface protein expression, cytokine production and leukocyte cross-talk potential. Further, sfRA-FLS are comparable to tissue-derived FLS in their capabilities to invade cartilage at implantation sites but also spread tissue destruction to a distant site. Collectively, sfRA-FLS can serve as a an easy-to-obtain source of pathological sublining FLS in RA.

HIV infects long-lived CD4 memory T cells, establishing a latent viral reservoir that necessitates lifelong antiretroviral therapy (ART). How this reservoir is formed so quickly after infection remains unclear. We now show the innate inflammatory response to HIV infection results in CCL2 chemokine release, leading to recruitment of cells expressing the CCR2 receptor, including a subset of central memory CD4 T cells. Supporting a role for the CCL2/CCR2 axis in rapid reservoir formation, we find (i) treatment of humanized mice with anti-CCL2 antibodies during early HIV infection decreases reservoir seeding and preserves CCR2/5(+) cells and (ii) CCR2/5(+) cells from the blood of HIV-infected individuals on long-term ART contain significantly more integrated provirus than CCR2/5-negative memory or naive cells. Together, these studies support a model where the host's innate inflammatory response to HIV infection, including CCL2 production, leads to the recruitment of CCR2/5(+) central memory CD4 T cells to zones of virus-associated inflammation, likely contributing to rapid formation of the latent HIV reservoir. IMPORTANCE There are currently over 35 million people living with HIV worldwide, and we still have no vaccine or scalable cure. One of the difficulties with HIV is its ability to rapidly establish a viral reservoir in lymphoid tissues that allows it to elude antivirals and the immune system. Thus, it is important to understand how HIV accomplishes this so we can develop preventive strategies. Our current results show that an early inflammatory response to HIV infection includes production of the chemokine CCL2, which recruits a unique subset of CCR2/5(+) CD4(+) T cells that become infected and form a significant reservoir for latent infection. Furthermore, we show that blockade of CCL2 in humanized mice significantly reduces persistent HIV infection. This information is relevant to the development of therapeutics to prevent and/or treat chronic HIV infections.

The chemokine receptor CCR5 is expressed on multiple cell types, including macrophages, dendritic cells, and T cells, and is the major co-receptor used during HIV transmission. Using a standard αCD3/CD28in vitrostimulation protocol to render CD4+ T cells from PBMCs permissive to HIV infection, we discovered that the percentage of CCR5+T cells was significantly elevated in CD4+ T cells when stimulated in the presence of peripheral blood mononuclear cells (PBMCs) as compared to when stimulated as purified CD4+ T cells. This indicated that environmental factors unique to the T-PBMCs condition affect surface expression of CCR5 on CD4+ T cells. Conditioned media from αCD3/CD28-stimulated PBMCs induced CCR5 expression in cultures of unstimulated cells. Cytokine profile analysis of these media suggests IL-12 as an inducer of CCR5 expression. Mass cytometric analysis showed that stimulated T-PBMCs exhibited a uniquely activated phenotype compared to T-Pure. In line with increased CCR5 expression and activation status in stimulated T-PBMCs, CD4+ T cells from these cultures were more susceptible to infection by CCR5-tropic HIV-1 as compared with T-Pure cells. These results suggest that in order to increaseex vivoinfection rates of blood-derived CD4+ T cells, standard stimulation protocols used in HIV infection studies should implement T-PBMCs or purified CD4+ T cells should be supplemented with IL-12.

The majority of HIV infections are established through the genital or rectal mucosa. Fibroblasts are abundant in these tissues, and although not susceptible to infection, can potently enhance HIV infection of CD4+ T cells. Hyaluronic acid (HA) is a major component of the extracellular matrix of fibroblasts, and its levels are influenced by the inflammatory state of the tissue. Since inflammation is known to facilitate HIV sexual transmission, we investigated the role of HA in genital mucosal fibroblast-mediated enhancement of HIV infection. Depletion of HA by CRISPR-Cas9 in primary foreskin fibroblasts augmented the ability of the fibroblasts to increase HIV infection of CD4+ T cells. This amplified enhancement required direct contact between the fibroblasts and CD4+ T cells, and could be attributed to both increased rates of trans-infection and the increased ability of HA-deficient fibroblasts to push CD4+ T cells into a state of higher permissivity to infection. This HIV-permissive state was characterized by differential expression of genes associated with regulation of cell metabolism and death. Our results suggest that conditions resulting in diminished cell-surface HA on fibroblasts, such as genital inflammation, can promote HIV transmission by conditioning CD4+ T cells toward a state more vulnerable to infection by HIV.

Background:Fibroblast-like synoviocytes (FLS) are central cellular components in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). Pathological subsets of FLS have been identified from synovial tissue. However, the synovial tissue obtained from arthroplasty procedures is acquired at late disease stages and the cellular yield obtained from synovial tissue biopsies is fairly low. Collectively, challenging the robustness of human RAin vivoandin vitromodels. FLS obtained from the synovial fluid (SF-FLS) are proposed as an alternative source of FLS, but a detailed phenotypical and functional characterization of FLS subsets from the synovial fluid has not been performed.Objectives:The aim of this study was to determine the phenotypical and functional characteristics of synovial fluid-derived fibroblast-like synoviocytes in rheumatoid arthritis.Methods:In the present study, paired peripheral blood mononuclear cells (PBMC) and SF-FLS from patients with RA were obtained (n=7). FLS were isolated from the synovial fluid by a strict trypsinization protocol1and their cellular characteristics and functionality were evaluated at passage 4. Monocultures (SF-FLS) and autologous co-cultures (SF-FLS and PBMC) were established from five patients with RA and subsequently evaluated by flow cytometry, Western blotting and multiplex immunoassays. Human cartilage-sponges (n=3) with SF-FLS and without SF-FLS (n=3) were co-implanted subcutaneously in SCID mice (n=15), mice with only cell-free human cartilage-sponges were used as controls (n=12). After 45 days, the implants were evaluated using stained sections to determine the SF-FLS invasion score based on perichondrocytic cartilage degradation. Data are expressed as median (25-75 percentile). P-values Results:The homogeneous subpopulations of FLS, isolated from the synovial fluid, were negative for CD34 and CD45 [98.9%, (97.5-99.7]) and positive for Thy-1 and PDPN [94.6%, (79.9-97.4]). Without stimulation, RA SF-FLS showed high and comparable levels of NFκB related pathway proteins and secreted multiple pro-inflammatory cytokines and chemokines dominated by IL-6 [2648 pg/mL, (1327-6116)] and MCP-1 [2458 pg/mL, (692-8719)]. SF-FLS increased their ICAM-1 and HLA-DR expression after encountering autologous PBMCs (pin vivowas at primary site, [1.6, (1.3-1.7)] and contralateral implantation site [1.5, (1.1-2.2)]. The invasion score of the human SF-FLS-containing implants both at primary and contralateral site were significantly higher compared with cartilage-sponges evaluated from SF-FLS-free control mice (pConclusion:This phenotypical and functional characterization of SF-FLS, acquired and activated at the site of pathology, lays a foundation for establishingin vivoandin vitroFLS models. These FLS models will be beneficial in our understanding of the role of this cellular subset in arthritis and for characterization of drugs specifically targeting this pathological RA FLS subset.References:[1]Nielsen M. A. et al. Responses to Cytokine Inhibitors Associated with Cellular Composition in Models of Immune-Mediated Inflammatory Arthritis. ACR Open Rheumatology, 2(1):3-10.http://doi.org/10.1002/acr2.11094Disclosure of Interests:Ditte Køster: None declared, Johanne Hovgaard Egedal: None declared, Malene Hvid: None declared, Martin Roelsgaard Jakobsen: None declared, Ulf Müller-Ladner Speakers bureau: Biogen, Bent Deleuran: None declared, Tue Wenzel Kragstrup Shareholder of: iBio Tech ApS, Consultant of: Bristol-Myers Squibb, Speakers bureau: TWK has engaged in educational activities talking about immunology in rheumatic diseases receiving speaking fees from Pfizer, Bristol-Myers Squibb, Eli Lilly, Novartis, and UCB., Elena Neumann: None declared, Morten Aagaard Nielsen: None declared

Innate immune activation by macrophages is an essential part of host defence against infection. Cytosolic recognition of microbial DNA in macrophages leads to induction of interferons and cytokines through activation of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING). Other host factors, including interferon-gamma inducible factor 16 (IFI16), have been proposed to contribute to immune activation by DNA. However, their relation to the cGAS-STING pathway is not clear. Here, we show that IFI16 functions in the cGAS-STING pathway on two distinct levels. Depletion of IFI16 in macrophages impairs cGAMP production on DNA stimulation, whereas overexpression of IFI16 amplifies the function of cGAS. Furthermore, IFI16 is vital for the downstream signalling stimulated by cGAMP, facilitating recruitment and activation of TANK-binding kinase 1 in STING complex. Collectively, our results suggest that IFI16 is essential for efficient sensing and signalling upon DNA challenge in macrophages to promote interferons and antiviral responses., The role of IFI16 as a DNA sensor is highly controversial. With support from a Nature Communications back-to-back publication from Almine et al. the authors here provide functional evidence that IFI16 is required for DNA sensing via the cGAS-STING pathway in human macrophages.

Seeding of the latent HIV reservoir occurs quickly within hours to a few days after initial viral infection. It is the persistence of virus within this reservoir that thwarts an HIV cure and necessitates life-long antiretroviral therapy (ART). Central memory CD4 T cells harboring latent HIV proviruses form a major part of this reservoir. How the virus establishes latency so rapidly within this subset of CD4 T cells remains unknown. We find that HIV infection leads to rapid production of the CCL2 chemokine in human lymphoid CD4 T cells. Cas9RNP-mediated depletion of IFI16 and STING genes significantly reduces production of CCL2 occurring in response to HIV. IFI16 was previously implicated as the key sensor triggering CD4 T cell pyroptosis. Thus, infection may also stimulate a second pathway of IFI16-mediated signaling involving STING culminating in CCL2 production. CCL2 functions a potent chemoattractant for cells expressing CCR2. Lymphoid CCR2+ CD4 T cells express the CCR5 co-receptor for HIV and markers of central memory. Additionally, these cells express many markers previously associated with the latent HIV reservoir. CCR2+ CD4 T cells are highly permissive to latent and productive infection by both R5 and X4-tropic viruses. When CCR2+ CD4 T cells are purified from HIV-infected individuals on suppressive ART, these cells are enriched up to 75-fold for HIV proviral DNA compared to naïve cells (n=10, p=0.0078). Together, our findings support a model where HIV infection triggers the production of CCL2, leading to directed recruitment and infection of CCR2+ CD4 central memory T cells. Thus, HIV hijacks a component of the innate immune response to rapidly recruit precisely those target cells that ensure its long term persistence.