Interaction of Type 1 Porcine Reproductive and Respiratory Syndrome Virus With In Vitro Derived Conventional Dendritic Cells

The present study delineates the interaction of a typical PRRSV1.1 isolate 3267 (moderate virulence) with in vitro derived pig conventional dendritic cells, cDC1, cDC2, and a CD14+ population (designated as CD14+ DCs). cDC1 and cDC2 were not susceptible to 3267 infection, but a fraction of CD14+ DCs were infected. After exposure to the virus, all three DC types remained immature as determined by no increase of maturation molecules (MHC-I, MHC-II, CD80/86, CCR7), no release of cytokines, no modification of antigen presentation abilities, and no alteration of endocytic/phagocytic capabilities. However, when infected MARC-145 cells were used as a source of viral antigens, cDC2 and CD14+ DCs showed a significant increase in the expression of maturation molecules and substantial release of cytokines, notably IL-12/IL-23p40 (by both DC types) and IL-10 (by CD14+ DCs). To address the impact of PRRSV1 3267 on TLR3- and TLR7-mediated activation, cDC1, cDC2, and CD14+ DCs were inoculated by the virus (live or UV-inactivated) for 6 h prior to or simultaneously with the addition of poly I:C (TLR3 ligand) or gardiquimod (TLR7 ligand; not used for cDC1). Compared with using TLR ligand alone, combination with the virus did not result in any alteration to the maturation markers on all DC types but changed the cytokine response to either TLR3 or TLR7 ligand. Pre-exposure of cDC2 or CD14+ DCs to the live virus resulted in an increased production of IFN-α upon poly I:C stimulation, while pre-exposure to UV-inactivated virus tended to enhance the release of IL-10 upon gardiquimod stimulation. Simultaneous addition of the live virus and the TLR ligand either had no effect (mainly in cDC2) or impaired most of the cytokine release after gardiquimod stimulation (in CD14+ DCs). When used as antigen presenting cells, cDC2 pre-inoculated by the live virus before addition of gardiquimod impaired the proliferation of CD4–CD8– T cells. In the case of CD14+ DCs, pre-exposure to the live virus or simultaneously added with TLR3 or TLR7 ligand largely decreased the proliferation of CD4–CD8+ and CD4–CD8+ T-cell subsets. For cDC1, no significant changes were observed in cytokine responses or T-cell proliferation after poly I:C stimulation. Of note, cDC1 had a short life during in vitro culturing, for which the results obtained might be biased. Overall, exposure to PRRSV1 did not induce maturation of cDC1, cDC2, or CD14+ DCs, but modified TLR3 and TLR7-associated responses (except for cDC1), which may affect the development of adaptive immunity during PRRSV1 infection. Moreover, the sensing of infected cells was different from that of the free virus.

CD200R1 and CD200R1L expression is regulated during B cell development in swine and modulates the Ig production in response to the TLR7 ligand imiquimoid

The CD200R family comprises a group of paired receptors that can modulate the activation of immune cells. They are expressed both on myeloid cells and lymphocyte subsets. Here we report that the expression of these receptors on porcine B cells is tightly regulated, being mainly expressed on mature cells. The expression of the inhibitory receptors CD200R1 and/or its splicing variant CD200R1X2, either in combination or not with the activating receptor CD200R1L, is upregulated in sIgM+ effector/memory cells, and tends to decline thereafter as these cells progress to plasmablasts or switch the Ig isotype. sIgM+ naïve and primed cells only express, by contrast, the CD200R1X2 receptor. B-1 like cells also express CD200R1 isoforms, either alone or in combination with CD200R1L. Treatment of peripheral blood mononuclear cells with a monoclonal antibody specific for inhibitory receptors, enhances the IgM and IgG production induced by TLR7 stimulation suggesting a modulatory role of B cell functions of these receptors.

Oral administration of PEGylated TLR7 ligand ameliorates alcohol-associated liver disease via the induction of IL-22

Effective therapies for alcohol-associated liver disease (ALD) are limited; therefore, the discovery of new therapeutic agents is greatly warranted. Toll-like receptor 7 (TLR7) is a pattern recognition receptor for single-stranded RNA, and its activation prevents liver fibrosis. We examined liver and intestinal damage inTlr7−/−mice to determine the role of TLR7 in ALD pathogenesis. In an alcoholic hepatitis (AH) mouse model, hepatic steatosis, injury, and inflammation were induced by chronic binge ethanol feeding in mice, andTlr7deficiency exacerbated these effects. Because these results demonstrated that endogenous TLR7 signaling activation is protective in the AH mouse model, we hypothesized that TLR7 activation may be an effective therapeutic strategy for ALD. Therefore, we investigated the therapeutic effect of TLR7 agonistic agent, 1Z1, in the AH mouse model. Oral administration of 1Z1 was well tolerated and prevented intestinal barrier disruption and bacterial translocation, which thus suppressed ethanol-induced hepatic injury, steatosis, and inflammation. Furthermore, 1Z1 treatment up-regulated the expression of antimicrobial peptides, Reg3b and Reg3g, in the intestinal epithelium, which modulated the microbiome by decreasing and increasing the amount ofBacteroidesandLactobacillus,respectively. Additionally, 1Z1 up-regulated intestinal interleukin (IL)-22 expression. IL-22 deficiency abolished the protective effects of 1Z1 in ethanol-induced liver and intestinal damage, suggesting intestinal IL-22 as a crucial mediator for 1Z1-mediated protection in the AH mouse model. Collectively, our results indicate that TLR7 signaling exerts protective effects in the AH mouse model and that a TLR7 ligand, 1Z1, holds therapeutic potential for the treatment of AH.

Treating ApoE−/− mice with Toll-like receptor 7 ligand lead to expansion of marginal zone B cells, reduced plasma cholesterol and decreased atherosclerosis

Background The interplay between innate and adaptive immunity is involved in the cause of the life-threatening events in atherosclerosis, such as myocardial infarction and stroke. We have previously shown that higher levels of Toll-like receptor 7 (TLR7) expression in human atherosclerotic lesions are correlated to better patient outcome, emphasizing the role of the innate pattern recognizing receptors in the pathogenesis of atherosclerosis. Purpose Firstly, to investigate if activation of the innate immune receptor TLR7 in vivo in experimental atherosclerosis is a potential way to reduce disease and secondly to identify pathways upon TLR7 ligation treatment. Methods Apolipoprotein E (ApoE−/−) deficient mice with established disease were injected intraperitoneal with a TLR7 ligand. Local effects were evaluated by characterization of atherosclerotic lesions. Systemic effects of the treatment were investigated by immune composition analysis in the spleen and by plasma measurements Results In vivo treatment of ApoE−/−mice with TLR7 ligand arrested atherosclerotic lesion development in the aortic root. Moreover, in the lesions of the treated mice we detected decrease in necrotic area and decrease in accumulation of apoptotic cells. In the spleen we detected an expansion of marginal zone B cells accompanied with an increase in IgM antibodies against oxidized low-density lipoprotein (oxLDL). The treated mice had reduced plasma cholesterol levels. Conclusions In vivo administration of TLR7 ligand ameliorated the atherosclerotic burden in old ApoE−/− mice. Our findings indicate that TLR7 ligation is involved in control of inflammatory responses in atherosclerosis, suggesting that TLR7 is a potential therapeutic target. Further analysis is needed in order to elucidate the underlying mechanisms by which TLR7 exerts its protective role. Funding Acknowledgement Type of funding source: Private grant(s) and/or Sponsorship. Main funding source(s): Swedish Heart-Lung Foundation

TLR7 Expression Is Associated with M2 Macrophage Subset in Calcific Aortic Valve Stenosis

Calcific aortic valve stenosis (CAVS) is a common age-related disease characterized by active calcification of the leaflets of the aortic valve. How innate immune cells are involved in disease pathogenesis is not clear. In this study we investigate the role of the pattern recognition receptor Toll-like receptor 7 (TLR7) in CAVS, especially in relation to macrophage subtype. Human aortic valves were used for mRNA expression analysis, immunofluorescence staining, or ex vivo tissue assays. Response to TLR7 agonist in primary macrophages and valvular interstitial cells (VICs) were investigated in vitro. In the aortic valve, TLR7 correlated with M2 macrophage markers on mRNA levels. Expression was higher in the calcified part compared with the intermediate and healthy parts. TLR7+ cells were co-stained with M2-type macrophage receptors CD163 and CD206. Ex vivo stimulation of valve tissue with the TLR7 ligand imiquimod significantly increased secretion of IL-10, TNF-α, and GM-CSF. Primary macrophages responded to imiquimod with increased secretion of IL-10 while isolated VICs did not respond. In summary, in human aortic valves TLR7 expression is associated with M2 macrophages markers. Ex vivo tissue challenge with TLR7 ligand led to secretion of immunomodulatory cytokine IL-10. These results connect TLR7 activation in CAVS to reduced inflammation and improved clearance.

pDC Activation by TLR7/8 Ligand CL097 Compared to TLR7 Ligand IMQ or TLR9 Ligand CpG

Plasmacytoid dendritic cells (pDCs) express high levels of the toll-like receptors (TLRs) TLR7 and TLR9. In response to TLR7 and TLR9 ligands, pDCs are primary producers of type I interferons. Our previous study demonstrated that pDCs activated by the TLR7 ligand imiquimod (IMQ) and the TLR9 ligand CpG A can kill breast cancer cells in vitro and inhibit tumor growth in vivo. Moreover, we observed a distinctive morphological, phenotypic change in pDCs after activation by IMQ and CpG A. However, the effect of other TLR7 and TLR9 ligands on pDCs remains less understood. In this study, we treat pDCs with the TLR7 ligand IMQ, TLR7/8 ligands (CL097 and CL075), and three TLR9 ligands (different types of CpGs). The size of pDCs increased significantly after activation by TLR7, or TLR7/8 ligands. TLR7, TLR7/8, and TLR9 ligands similarly modulated cytokine release, as well as protein expression of pDC markers, costimulatory molecules, and cytotoxic molecules. Interestingly, TLR7/8 ligands, especially CL097, induced stronger responses. These results are relevant to the further study of the role and mechanism of pDC-induced antitumor effects and may aid in the development of a new strategy for future tumor immunotherapy.