Depletion Of Plasmacytoid Dendritic Cells Research Articles

Intestinal immunological events of acute and resolved SARS-CoV-2 infection in non-human primates

SARS-CoV-2 infection has been associated with intestinal mucosal barrier damage, leading to microbial and endotoxin translocation, heightened inflammatory responses, and aggravated disease outcomes. This study aimed to investigate the immunological mechanisms associated with impaired intestinal barrier function. We conducted a comprehensive analysis of gut damage and inflammation markers and phenotypic characterization of myeloid and lymphoid populations in the ileum and colon of SARS-CoV-2-exposed macaques during both the acute and resolved infection phases. Our findings revealed a significant accumulation of terminally differentiated and activated CD4+ and CD8+ T cells, along with memory B cells, within the gastrointestinal tract up to 43 days after exposure to SARS-CoV-2. This robust infection-induced immune response was accompanied by a notable depletion of plasmacytoid dendritic cells, myeloid dendritic cells, and macrophages, particularly affecting the colon during the resolved infection phase. Additionally, we identified a population of CX3CR1Low inflammatory macrophages associated with intestinal damage during active viral replication. Elevated levels of immune activation and gut damage markers, and perturbation of macrophage homeostasis, persisted even after the resolution of the infection, suggesting potential long-term clinical sequelae. These findings enhance our understanding of gastrointestinal immune pathology following SARS-CoV-2 infection and provide valuable information for developing and testing medical countermeasures.

Direct and Indirect type I Interferon signaling regulates Eomes-driven Th1-responses against Influenza Virus

Abstract In the absence of Th1 master regulator, the T-box transcription factor, T-bet, CD4 T cells primed by Influenza A Virus (IAV) infection, gain Th17 attributes while still retaining robust Th1 functionality. We recently found the transcription factor Eomesodermin (Eomes), to regulate this residual Th1 identity of T-bet −/−CD4 T cells. However, it is unclear how Eomes is regulated in CD4 T cells, especially during antiviral responses. Here we find type I interferons to be potent inducers of Eomes but not of T-bet in naïve antigen specific CD4 T cells stimulated in vitro. Furthermore, blocking type I IFN signaling to T-bet −/−CD4 T cells in IAV infected WT host mice, reduces Eomes expression and drives functional shifts mirroring T-bet −/−Eomes −/−cells, including significantly reduced IFNγ production and stronger Th17 identity. To test the impact of direct type I IFN signals on Eomes-driven Th1 identity of T-bet −/−CD4 T cells, we compared the responses of T-bet −/−CD4 T cells versus T-bet −/−cells also deficient for type I IFN receptor. While the Th1 attributes of the double-deficient cells were compromised compared to T-bet −/−cells, T-bet −/−cells responding in hosts treated to block systemic type I IFN signaling were the most impacted. These results indicate a strong role for indirect type I IFN signals in regulating Eomes-dependent functions during antiviral CD4 T cells responses. Finally, we find depletion of plasmacytoid dendritic cells in IAV infected host mice, a major source of type I IFN, does not impact T-bet −/−CD4 T cell response. Together, these results identify an important axis controlling the expression of Eomes in antiviral CD4 T cells that may in the future be harnessed to fine-tune effector and memory attributes primed by vaccination Supported by grants from NIH (R01 AI67994 and R21 AI146647)

Depletion of plasmacytoid dendritic cells and HIF-1α inhibition enhance immunotherapy against hepatocellular carcinoma

Abstract Hepatocellular Carcinoma (HCC) is one of the deadliest forms of cancer. Recent years have seen significant advances for HCC treatment, but up to 70% of patients remain at high risk for recurrence necessitating the urgent need for more effective therapies. Plasmacytoid dendritic cells (pDCs) heavily infiltrate liver tumor tissues, contribute to an immunosuppressive tumor microenvironment (TEM), and promote HCC growth. Furthermore, hypoxia often occurs in HCC tumor tissues and plays the central role of an immunosuppressive TME. A hypoxic TME enhances pDC recruitment into tumor tissues via the upregulation of hypoxia-inducible factor 1α (HIF-1α). Here we show that the distribution of pDCs does not co-localized with hypoxic areas in HCC. In the immunocompetent HCC mouse model, depletion of pDCs using the antibody or HIF-1α inhibitor echinomycin significantly suppressed tumor growth. The combination of pDC depletion and HIF-1α inhibition suppressed HCC growth more effectively. Both treatments reshaped the TME with changes of the cytokine profile and increased tumor-infiltrating CD8 T cells. Reduced basic helix-loop-helix transcription factor E2-2 expression by treatments abrogated protumor functions of pDCs in HCC resulting in reactivating tumor-reactive CD8 T cells. This study demonstrates that targeting both pDCs and HIF-1α may serve as more effective immunotherapy for HCC.

Comparison of cell type annotation algorithms for revealing immune response of COVID-19

When analyzing scRNA-seq data with clustering algorithms, annotating the clusters with cell types is an essential step toward biological interpretation of the data. Annotations can be performed manually using known cell type marker genes. Annotations can also be automated using knowledge-driven or data-driven machine learning algorithms. Majority of cell type annotation algorithms are designed to predict cell types for individual cells in a new dataset. Since biological interpretation of scRNA-seq data is often made on cell clusters rather than individual cells, several algorithms have been developed to annotate cell clusters. In this study, we compared five cell type annotation algorithms, Azimuth, SingleR, Garnett, scCATCH, and SCSA, which cover the spectrum of knowledge-driven and data-driven approaches to annotate either individual cells or cell clusters. We applied these five algorithms to two scRNA-seq datasets of peripheral blood mononuclear cells (PBMC) samples from COVID-19 patients and healthy controls, and evaluated their annotation performance. From this comparison, we observed that methods for annotating individual cells outperformed methods for annotation cell clusters. We applied the cell-based annotation algorithm Azimuth to the two scRNA-seq datasets to examine the immune response during COVID-19 infection. Both datasets presented significant depletion of plasmacytoid dendritic cells (pDCs), where differential expression in this cell type and pathway analysis revealed strong activation of type I interferon signaling pathway in response to the infection.

Interferon-α exerts proinflammatory properties in experimental radiation-induced esophagitis: Possible involvement of plasmacytoid dendritic cells

AimsRadiation-induced esophagitis, experienced during radiation therapy for lung cancer and head and neck cancer, is a major dose-limiting side effect of the treatment. This study aimed to elucidate the role of interferon-α (IFN-α) in radiation-induced esophagitis. Main methodsC57BL/6 mice were exposed to 10 and 25Gy of single thoracic irradiation. Esophageal mucosal damage and inflammatory reactions were assessed for 5 days after irradiation. Key findingsIrradiation induced esophagitis, characterized by reduction in the thickness of epithelial layer, upregulation of proinflammatory cytokines and chemokines, infiltration of inflammatory cells into the esophageal mucosa, and apoptosis of epithelial cells. Irradiation upregulated the level of gene expression for IFN-α in the esophageal tissue, and the neutralizing antibody against IFN-α ameliorated radiation-induced esophageal mucosal damage, while administration of IFN-α receptor agonist (RO8191) had an inverse effect. Depletion of plasmacytoid dendritic cells (pDCs) by anti-CD317 antibody or pharmacological inactivation with bortezomib suppressed radiation-induced mucosal inflammation and damage, accompanied by decrease in IFN-α expression level. SignificanceThese findings suggest that IFN-α and pDCs exert proinflammatory properties in the pathophysiology of radiation-induced esophagitis.

Dysregulation of Type 1 Interferon Signaling in Systemic Sclerosis: a Promising Therapeutic Target?

There are several lines of evidence at the genetic and gene expression levels linking type I interferon (IFN) activation to systemic sclerosis (SSc) pathogenesis. Herein, we summarize the potential role of type I IFN signaling components as therapeutic targets. All type I IFN cytokines signal through the interferon-α/β receptor (IFNAR). Early phase studies indicate that anifrolumab (a human monoclonal antibody against IFNAR subunit 1) has an acceptable safety profile and can attenuate transforming growth factor beta (TGF-β)-mediated fibrosis in SSc skin, supporting its further clinical development. Janus kinase (JAK) signaling pathways are downstream from IFNAR. Building on their efficacy in hereditary interferonopathies, JAK inhibitors have the potential to block the deleterious IFN and other profibrotic cytokine activation in SSc and are promising drug targets. Moreover, interferon regulator factor (IRF) 5, 7, and 8 have been linked to the profibrotic response in SSc preclinical studies, underscoring their potential as therapeutic targets. Lastly, depletion of plasmacytoid dendritic cells (pDCs) attenuates the IFN activation and fibrotic response in vitro and murine model experiments and can be studied as a viable drug target in future clinical studies. There is increasing evidence linking the prominent type I IFN activation to the observed exaggerated fibrotic response in SSc. Key components of type I IFN signaling are druggable therapeutic targets that can be pursued in future randomized clinical trials, in order to develop more effective therapeutic options for SSc.

Bleomycin-Induced Lung Injury Increases Resistance to Influenza Virus Infection in a Type I Interferon-Dependent Manner.

Acute lung injury (ALI) results in acute respiratory disease that causes fatal respiratory diseases; however, little is known about the incidence of influenza infection in ALI. Using a ALI-mouse model, we investigated the pro-inflammatory cytokine response to ALI and influenza infection. Mice treated with bleomycin (BLM), which induces ALI, were more resistant to influenza virus infection and exhibited higher levels of type I interferon (IFN-I) transcription during the early infection period than that in PBS-treated control mice. BLM-treated mice also exhibited a lower viral burden, reduced pro-inflammatory cytokine production, and neutrophil levels. In contrast, BLM-treated IFN-I receptor 1 (IFNAR1)-knockout mice failed to show this attenuated phenotype, indicating that IFN-I is key to the antiviral response in ALI-induced mice. The STING/TBK1/IRF3 pathway was found to be involved in IFN-I production and the establishment of an antiviral environment in the lung. The depletion of plasmacytoid dendritic cells (pDCs) reduced the effect of BLM treatment against influenza virus infection, suggesting that pDCs are the major source of IFN-I and are crucial for defense against viral infection in BLM-induced lung injury. Overall, this study showed that BLM-mediated ALI in mice induced the release of double-stranded DNA, which in turn potentiated IFN-I-dependent pulmonary viral resistance by activating the STING/TBK1/IRF3 pathway in association with pDCs.

A dynamic COVID-19 immune signature includes associations with poor prognosis.

Improved understanding and management of COVID-19, a potentially life-threatening disease, could greatly reduce the threat posed by its etiologic agent, SARS-CoV-2. Toward this end, we have identified a core peripheral blood immune signature across 63 hospital-treated patients with COVID-19 who were otherwise highly heterogeneous. The signature includes discrete changes in B and myelomonocytic cell composition, profoundly altered T cell phenotypes, selective cytokine/chemokine upregulation and SARS-CoV-2-specific antibodies. Some signature traits identify links with other settings of immunoprotection and immunopathology; others, including basophil and plasmacytoid dendritic cell depletion, correlate strongly with disease severity; while a third set of traits, including a triad of IP-10, interleukin-10 and interleukin-6, anticipate subsequent clinical progression. Hence, contingent upon independent validation in other COVID-19 cohorts, individual traits within this signature may collectively and individually guide treatment options; offer insights into COVID-19 pathogenesis; and aid early, risk-based patient stratification that is particularly beneficial in phasic diseases such as COVID-19.

Graft-Versus-Host Disease Causes the Failure of Donor Hematopoietic Progenitor Cells to Reconstitute Plasmacytoid Dendritic Cells That Promote Tolerance of Donor T Cells Against the Host

Promoting donor T cell tolerance to host non-hematopoietic tissues remains the ultimate therapeutic goal in allogeneic hematopoietic stem cell transplantation (allo-HSCT). Dendritic cells (DCs) play dual functions in regulating alloimmunity. DCs can elicit alloreactive T cell responses to mediate graft-versus-host disease (GVHD), but are also implicated in reducing GVHD. In patients, the depletion of plasmacytoid DCs (pDCs) from donor BM grafts resulted in GVHD acceleration. On the other hand, acute GVHD causes complete failure of donor pDC reconstitution after allo-HSCT, and low levels of donor pDC correlate with significantly increased GVHD severity. Thus, the impairment of pDC reconstitution by GVHD may be responsible for the dysfunctional immune regulation. Delineation of the mechanism involved may allow therapeutic intervention to reduce GVHD and improve the efficacy of allo-HSCT. In this study, we demonstrate that alloreactive T cells produce GM-CSF to impair reconstitution of donor pDCs by inhibiting Flt3 expression and its-regulated transcription programs in DC progenitor cells. Using murine GVHD model, we confirmed GVHD severely impaired reconstitution of both donor pDCs and conventional DCs (cDCs). Adoptive transfer of donor-type pDCs rather than cDCs prevented the occurrence of severe GVHD in mice, suggesting donor pDC reconstitution is important to restore tolerance of donor T cells against host tissues. Flt3 is required to induce pDC production through a successive differentiation pathway: HSC → multiple potential progenitors (MPP) → common DC progenitors (CDP) → precursor DCs (pre-DCs). GVHD mice produced significantly less MPP, CDP and pre-DCs compared to normal donor mice and allogeneic mice receiving T cell-depleted BM. Ex vivo culture with Flt3 ligand (Flt3L) showed that those MPP and CDP derived from GVHD mice dramatically decreased the capacity to produce pDCs. Thus, GVHD not only causes decreased numbers of MPP and CDP but also their intrinsic defect in producing pDCs. While both MPP and CDP gave rise to similar numbers of pDCs within 3 days of culture with Flt3L, MPP produced 40-fold more pDCs than CDP by day 9 of culture. This indicates the impairment in GVHD MPP may have much more profound long-term impact on pDC reconstitution than that in CDP. Based on surface expression of Flt3, normal MPP contained two subsets: CD135high MPP and CD135mod MPP. CD135high MPP produced 4-fold more pDCs than CD135mod MPP. As compared to CD135mod MPP, CD135high MPP expressed lower levels of Ink4 family genes, which are cyclin-dependent inhibitors restraining cell proliferation and survival, suggesting that CD135high MPP represent earlier stage differentiated progenitors with greater proliferative capacity. Intriguingly, although GVHD mice generated similar amount of CD135mod MPP as did normal mice, they failed to reconstitute highly proliferative CD135high MPP. Thus, the failure of donor pDC reconstitution may largely result from GVHD-mediated inhibition of CD135high MPP. Alloreactive T cells are known to produce high levels of effector molecules, such as IFN-γ, TNF-α, GM-CSF and other cytolytic molecules. We observed that GVHD effector T cells significantly reduced the production of pDCs from Flt3L-induced normal MPP. Blocking GM-CSF using neutralizing antibody but not other effector molecules markedly inhibited this repressive effect of GVHD T cells on pDC production. GM-CSF dose-dependently decreased the expression of Flt3 and its-regulated transcription factors Irf8 and Tcf4, which are important for development of functional pDCs. However, GM-CSF failed to inhibit the conversion of SiglecH+ pre-pDCs into pDCs. These data suggest that alloreactive T cells produce GM-CSF to block pDC reconstitution by targeting DC progenitors (e.g., MPP and CDP). Building on these findings, we established a novel optimized culture system to produce adequate numbers of SiglecH+ pre-pDCs. Adoptive transfer of these pre-pDCs prevented GVHD, leading to significantly improved overall survival of mice undergoing allo-HSCT. Our findings identify for the first time that selective restoration of donor pDCs early after allo-HSCT may represent an effective cellular therapy to prevent GVHD. Further delineation of the molecular pathway(s) involved in GVHD inhibition of DC progenitors may allow the development of novel approaches to circumvent mortality and morbidity associated with GVHD. Disclosures Zheng: Pfizer: Research Funding.

Plasmacytoid dendritic cell depletion modifies FoxP3+ T cell homeostasis and the clinical course of bacterial pneumonia in mice.

Plasmacytoid dendritic cells (pDC) are critical to antiviral defense because of their high production of type I IFNs; less is known regarding their functions in bacterial infection. Moreover, pDC are involved in immunomodulation. A stable pool of regulatory T cells (Treg) is crucial for maintaining immune homeostasis. However, interactions between pDC and Treg regarding the regulation of Treg homeostasis are understudied. By using BDCA2-DTR mice as a systemic pDC depletion model, we identified increased steady-state numbers of FoxP3+ T cells with an effector Treg-like phenotype in lungs, liver, and spleen tissues. During sublethal, pulmonary Klebsiella pneumoniae infection, pDC deficiency also elevated respiratory FoxP3+ T cell numbers. Additionally, the improvement in acute pneumonia survival until day 5 post infection was accompanied by impaired proinflammatory cytokine production. In contrast, pDC-depleted mice exhibited a delayed clinical recovery during the post-acute phase. Therefore, we assume that pDC act as immunomodulators supporting the rapid onset of immune response in a proinflammatory manner and regulate inflammation or tissue regeneration in the post-acute phase. In summary, pDC assist in FoxP3+ T cell homeostasis and the regulation of Klebsiella-pneumonia progression.

Immunome Single Cell Profiling Reveals T Cell Exhaustion with Upregulation of Checkpoint Inhibitors LAG3 and Tigit on Marrow Infiltrating T Lymphocytes in Daratumumab and IMiDs Resistant Patients

Background: The addition of immunomodulatory (IMiDs) drugs to monoclonal antibodies targeting the transmembrane glycoprotein CD38 has demonstrated very encouraging and durable responses in myeloma patients. It is believed that the synergistic effects observed with anti-CD38 antibodies and IMiDs are derived from their co-modulation of the host adaptive and innate immunity and therefore it is plausible to speculate that acquired resistance to daratumumab and IMiDs may be largely immune-mediated. The aim of the present study was to 1) interrogate at the single cell level the bone marrow immune repertoire of daratumumab sensitive and resistant patients, 2) identify cellular mediators of resistance to anti-CD38 antibodies and 3) define potential means to reinstate sensitivity to daratumumab and IMiDs.Methods and Results: Serial BM aspirates (n=44) were collected from patients treated with single agent daratumumab (MMY3012 trial) or daratumumab + pomalidomide (MM014 trial) prior to initiation of therapy, C3D1 and at relapse. Bone marrow mononuclear fractions were isolated through ficoll density gradients coupled with magnetic sorting of CD138pos and CD138neg cells. Unbiased mRNA profiling of BM CD138neg cells was performed by single-cell RNA-seq (scRNA-seq) using the GemCode system (10x Genomics). Paired-end sequencing was performed on Illumina NEXTseq and NOVAseq platforms. Cell Ranger Single and Seurat were used for sample de-multiplexing, barcode processing, single-cell 3′ gene counting and data analysis. Sequencing data were analyzed by principal component analysis (PCA), clustering with multi-sample batch correction and then visualized by t-distributed stochastic neighbor embedding (t-SNE) projection. Comparison of the single cell transcriptomes of CD138neg cells from responding patients pre- and post- treatment revealed that Daratumumab and Pomalidomide dramatically modify the immune cells composition (immunome) of the bone marrow niches leading to: 1) expansion of effector T cells (KLRG1high, GZMAhigh, CCL5high), 2) significant depletion of CD38high / FCGR3Ahigh NK cells with retained population of cytotoxic NK cells (CD27high, KLRB1high, NCR3high, GZMApos, PRF1pos), 3) depletion of FCGR3Ahigh / CD14low monocytes, 4) expansion of M1 inflammatory macrophages and depletion of plasmacytoid dendritic cells. Similar changes were seen in patients treated with single agent daratumumab (without IMiDs) however with a lesser expansion of effector T cells and in particular reduced marrow infiltrating inflammatory macrophages. In contrast, the immunome of daratumumab and pomalidomide resistant patients was characterized by a reduced central memory T cells (TCM), and a largely exhausted effector T cells populations that are CD28neg and expressing checkpoint inhibitors (LAG3and TIGIT significantly more than PDCD1) as well as high expression of TIM3 (HAVCR2) on marrow macrophages. Upregulation of LAG3 and TIGIT expression on T cells was also confirmed at the antigenic level by flow cytometry. Consistent with the non-bystander and suppressive effect of LAG3 and TIGIT on the function of effector T cells, activation (CD107a expression) and proliferation of LAGpos and/or TIGITpos sorted bone marrow T cells from resistant patients were significantly reduced in response to autologous myeloma cells stimulation or CD3/CD28 crosslinking. Lastly, a higher proportion and number of clonal T cell (through single cell TCR sequencing) was also observed in responding (≥ PR) vs non-responding (< PR) patients. Interrogation of the myeloma cells transcriptome, showed little to no loss of CD38 transcript at the time of acquired resistance with rather upregulation of complement inhibitory molecule CD59 and NFκB signature genes.Conclusion: A systematic unsupervised interrogation of the bone marrow immunome of daratumumab and IMiDs treated MM patients demonstrated a significant activation of adaptive and innate immunity in responding patients and revealed an expansion of exhausted T cells with upregulation of the checkpoint inhibitors LAG3 and TIGIT in resistant patients. Our findings warrant the exploration of LAG3- and/or TIGIT-blocking strategies as potential means to reinstate sensitivity to daratumumab and IMiDs in myeloma patients. DisclosuresNeri:Janssen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. McCulloch:Celgene: Honoraria; Takeda: Other: Travel expenses. Thakurta:Celgene Corporation: Employment, Equity Ownership. Bahlis:Celgene: Consultancy, Honoraria, Research Funding; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria, Research Funding.

Porcine B Cell Subset Responses to Toll-like Receptor Ligands.

Toll-like receptors (TLR) triggering of B cells are known to promote B cell expansion, differentiation of B cells into antibody-producing and memory cells, but the TLR responses of porcine B cells is poorly characterized. Therefore, this study investigated the response pattern of porcine B cell subsets to a large collection of TLR ligands and demonstrates that the TLR2 ligand Pam3Cys-SK4 and the TLR7/8 ligands gardiquimod and resiquimod are particularly efficient at inducing proliferation, CD25 and CCR7. This activation was also determined in B-cell subpopulations including a CD21+IgM+ subset, an IgG+ subset and two putative B1-like subsets, defined as CD21−IgMhighCD11R1+CD11c+CD14+ and CD21−IgMhigh CD11R1−CD11c+CD14− B cells. The latter two were larger and expressed higher levels of CD80/86 and spontaneous phospholipase C-γ2 phosphorylation. All porcine B-cell subsets were activated by TLR2, TLR7, and TLR9 ligands. Naïve and memory conventional B cells responded similar to TLR ligands. The CD11R1+ B1-like subset had the highest proliferative responses. While both B1-like subsets did not spontaneously secrete IgM, they were the only subsets to produce high level of TLR-induced IgM. Similar to polyclonal IgM responses, memory B cells were efficiently induced to produce specific antibodies by CpG oligodinucleotide, resiquimod, and to a weaker extend by Pam3Cys-SK4. Depletion of plasmacytoid dendritic cells (pDCs) enhanced TLR-induced antibodies. The same set of TLR ligands also induced CD40 on cDCs, pDCs, and monocytes with the exception of TLR4 ligand being unable to activate pDCs. Gardiquimod and resiquimod were particularly efficient at inducing CCR7 on pDCs. Porcine B cells expressed high levels of TLR7, but relatively little other TLR mRNA. Nevertheless, TLR2 on B cells was rapidly upregulated following stimulation, explaining the strong responses following stimulation. Subset-specific analysis of TLR expression demonstrated a comparable expression of TLR2, TLR7, and TLR9 in all B cell subsets, but TLR3 was restricted to B1-like cells, whereas TLR4 was only expressed on conventional B cells, although both at low levels. Altogether, our data describe porcine innate B1-like cells, and how different B cell subsets are involved in innate sensing.

HIV-1 infection depletes human CD34+CD38- hematopoietic progenitor cells via pDC-dependent mechanisms.

Chronic human immunodeficiency virus-1 (HIV-1) infection in patients leads to multi-lineage hematopoietic abnormalities or pancytopenia. The deficiency in hematopoietic progenitor cells (HPCs) induced by HIV-1 infection has been proposed, but the relevant mechanisms are poorly understood. We report here that both human CD34+CD38- early and CD34+CD38+ intermediate HPCs were maintained in the bone marrow (BM) of humanized mice. Chronic HIV-1 infection preferentially depleted CD34+CD38- early HPCs in the BM and reduced their proliferation potential in vivo in both HIV-1-infected patients and humanized mice, while CD34+CD38+ intermediate HSCs were relatively unaffected. Strikingly, depletion of plasmacytoid dendritic cells (pDCs) prevented human CD34+CD38- early HPCs from HIV-1 infection-induced depletion and functional impairment and restored the gene expression profile of purified CD34+ HPCs in humanized mice. These findings suggest that pDCs contribute to the early hematopoietic suppression induced by chronic HIV-1 infection and provide a novel therapeutic target for the hematopoiesis suppression in HIV-1 patients.

Association of ORMDL3 with rhinovirus-induced endoplasmic reticulum stress and type I Interferon responses in human leucocytes.

Children with risk alleles at the 17q21 genetic locus who wheeze during rhinovirus illnesses have a greatly increased likelihood of developing childhood asthma. In mice, overexpression of the 17q21 gene ORMDL3 leads to airway remodelling and hyperresponsiveness. However, the mechanisms by which ORMDL3 predisposes to asthma are unclear. Previous studies have suggested that ORMDL3 induces endoplasmic reticulum (ER) stress and production of the type I interferon (IFN)-regulated chemokine CXCL10. The purpose of this study was to determine the relationship between ORMDL3 and rhinovirus-induced ER stress and type I IFN in human leucocytes. ER stress was monitored by measuring HSPA5, CHOP and spliced XBP1 gene expression, and type I IFN by measuring IFNB1 (IFN-β) and CXCL10 expression in human cell lines and primary leucocytes following treatment with rhinovirus. Requirements for cell contact and specific cell type in ORMDL3 induction were examined by transwell assay and depletion experiments, respectively. Finally, the effects of 17q21 genotype on the expression of ORMDL3, IFNB1 and ER stress genes were assessed. THP-1 monocytes overexpressing ORMDL3 responded to rhinovirus with increased IFNB1 and HSPA5. Rhinovirus-induced ORMDL3 expression in primary leucocytes required cell-cell contact, and induction was suppressed by plasmacytoid dendritic cell depletion. The degree of rhinovirus-induced ORMDL3, HSPA5 and IFNB1 expression varied by leucocyte type and 17q21 genotype, with the highest expression of these genes in the asthma-associated genotype. Multiple lines of evidence support an association between higher ORMDL3 and increased rhinovirus-induced HSPA5 and type I IFN gene expression. These associations with ORMDL3 are cell type specific, with the most significant 17q21 genotype effects on ORMDL3 expression and HSPA5 induction evident in B cells. Together, these findings have implications for how the interaction of increased ORMDL3 and rhinovirus may predispose to asthma.

The regulatory dendritic cell marker C1q is a potent inhibitor of allergic inflammation.

The complement subunit C1q was recently identified as a marker for monocyte-derived regulatory dendritic cells supporting the differentiation of interleukin (IL)-10-secreting CD4+ T cells with a suppressive activity. Furthermore, C1q expression is upregulated in peripheral blood mononuclear cells of allergic patients in the course of successful allergen immunotherapy. Herein, we investigated a potential direct role of C1q in downregulating allergic inflammation. In mice with ovalbumin (OVA) or birch pollen (BP)-induced allergic asthma, C1q is as efficacious as dexamethasone to reduce both airway hyperresponsiveness (AHR), eosinophil, and ILC2 infiltrates in bronchoalveolar lavages, as well as allergen-specific T helper 2 cells in the lungs. Administration of C1q does not expand IL-10+/Foxp3+ regulatory T cells in the lungs, spleen, or in the blood. Depletion of plasmacytoid dendritic cells (pDCs) abrogates the capacity of C1q to reduce AHR and eosinophilic infiltrates in OVA-sensitized mice. Also C1q treatment inhibits the activation of human and mouse pDCs by CpGs, thereby demonstrating a critical role for pDCs in the anti-inflammatory activity of C1q. We conclude that regulatory dendritic cells can mediate a potent direct anti-inflammatory activity via the expression and/or secretion of molecules such as C1q, independently of their capacity to expand the pool of regulatory T cells.

Type I IFNs Regulate Inflammation, Vasculopathy, and Fibrosis in Chronic Cutaneous Graft-versus-Host Disease

Type I IFNs play a critical role in the immune response to viral infection and may also drive autoimmunity through modulation of monocyte maturation and promotion of autoreactive lymphocyte survival. Recent demonstrations of type I IFN gene signatures in autoimmune diseases, including scleroderma, led us to investigate the pathological role of IFNs in a preclinical model of sclerodermatous graft-versus-host disease. Using a neutralizing Ab against the type I IFN receptor IFNAR1, we observed a marked reduction in dermal inflammation, vasculopathy, and fibrosis compared with that seen in the presence of intact IFNAR1 signaling. The ameliorative effects of IFNAR1 blockade were restricted to the skin and were highly associated with inhibition of chronic vascular injury responses and not due to the inhibition of the T or B cell alloresponse. Inhibition of IFNAR1 normalized the overexpression of IFN-inducible genes in graft-versus-host disease skin and markedly reduced dermal IFN-α levels. Depletion of plasmacytoid dendritic cells, a major cellular source of type I IFNs, did not reduce the severity of fibrosis or type I IFN gene signature in the skin. Taken together, these studies demonstrate an important role for type I IFN in skin fibrosis, and they provide a rationale for IFNAR1 inhibition in scleroderma.

Chronic HIV-1 infection induces the depletion and functional impairment of group-3 innate lymphoid cells through plasmacytoid dendritic cells and type I interferon

Abstract Group 3 innate lymphoid cells (ILC3s) play a key role in promoting antibody production and gut inflammation and immunity; however, their characteristics and relevant regulatory mechanisms in chronic HIV-1 infection are poorly understood partially due to the lack of a robust model. We, here, reported that functional ILC3 cells are developed in all lymphoid organs in humanized mice with similar phenotypes to human counterparts. Importantly, chronic HIV-1 infection activated these tissue-resident ILC3 cells, and subsequently depleted them and preferentially impaired their IL-17a and IL-22 production in vivo of humanized mice, while anti-viral therapy efficiently reversed this alteration. HIV-1-induced depletion of ILC3s and their functional impairment could also be largely recovered by the depletion of plasmacytoid dendritic cells (pDCs) or in vivo of humanized mice and by the blockade of both interferon (IFN)-I and CD95/Fas pathway in vitro. Blockade of IFN-I also recovered IL-17a and IL-22 production in vivo of humanized mice with chronic HIV-1 infection. Additionally, we determined that HIV-1 infection induces CD95 expression on ILC3s via a pDC- and IFN-I–dependent mechanism that sensitizes ILC3s to undergo CD95/FasL-mediated apoptosis. Finally, ILC3 depletion was found to be associated with loss of gut mucosa barrier in HIV-1-infected patients. Thus, we conclude that chronic HIV-1 infection depletes ILC3s through pDC activation, induction of IFN-I, and CD95-mediated apoptosis.

Early, transient depletion of plasmacytoid dendritic cells ameliorates autoimmunity in a lupus model.

Plasmacytoid dendritic cells (pDCs) have long been implicated in the pathogenesis of lupus. However, this conclusion has been largely based on a correlative link between the copious production of IFN-α/β by pDCs and the IFN-α/β "signature" often seen in human lupus patients. The specific contribution of pDCs to disease in vivo has not been investigated in detail. For this reason, we generated a strain of BXSB lupus-prone mice in which pDCs can be selectively depleted in vivo. Early, transient ablation of pDCs before disease initiation resulted in reduced splenomegaly and lymphadenopathy, impaired expansion and activation of T and B cells, reduced antibodies against nuclear autoantigens and improved kidney pathology. Amelioration of pathology coincided with decreased transcription of IFN-α/β-induced genes in tissues. PDC depletion had an immediate impact on the activation of immune cells, and importantly, the beneficial effects on pathology were sustained even though pDCs later recovered, indicating an early pDC contribution to disease. Together, our findings demonstrate a critical function for pDCs during the IFN-α/β-dependent initiation of autoimmune lupus and point to pDCs as an attractive therapeutic target for the treatment of SLE.

Plasmacytoid dendritic cell depletion in DOCK8 deficiency: Rescue of severe herpetic infections with IFN-α 2b therapy

Plasmacytoid dendritic cell depletion in DOCK8 deficiency: Rescue of severe herpetic infections with IFN-α 2b therapy

Epicutaneous Application of Toll‐like Receptor 7 Agonists Leads to Systemic Autoimmunity in Wild‐Type Mice: A New Model of Systemic Lupus Erythematosus

To examine whether topical treatment of wild-type mice with Toll-like receptor 7 (TLR-7) agonists leads to lupus-like autoimmunity. Wild-type FVB/N, BALB/c, and C57BL/6 mice were treated with the topical TLR-7 agonist imiquimod or R848 administered to the ear 3 times weekly. During treatment, the mice were monitored for serum autoantibody and creatinine levels as well as histopathology of the kidneys, spleens, livers, hearts, and skin. Immunologic abnormalities were analyzed by immunohistochemistry, quantitative reverse transcription-polymerase chain reaction, and fluorescence-activated cell sorting. The role of plasmacytoid dendritic cells (PDCs) in the development of autoimmune disease was validated by in vivo treatment with an anti-PDC antibody. Diseased mice underwent ultraviolet B irradiation, to evaluate skin photosensitivity. The disease-causing effect of topical application of imiquimod was compared with that of systemic (intraperitoneal) administration. TLR-7- and TLR-9-deficient mice were used to validate the role of TLR-7. Wild-type mice of different genetic backgrounds developed systemic autoimmune disease following 4 weeks of topical treatment with imiquimod or R848, with elevated levels of autoantibodies to double-stranded DNA and multiple organ involvement, including glomerulonephritis, hepatitis, carditis, and photosensitivity. Expression of Ifna and Mx1, the interferon-α-stimulated gene, was up-regulated in the organs of imiquimod-treated mice. However, disease caused by intraperitoneal injection of imiquimod was less severe than that induced by topical application. In vivo depletion of PDCs by a specific antibody protected mice against the autoimmunity induced by topical administration of imiquimod, suggesting a role of PDCs. Furthermore, TLR-7-deficient mice, but not TLR-9-deficient mice, were protected against autoimmunity. This protocol provides a novel model of inducible systemic lupus erythematosus in wild-type mice and underscores the skin as the primary organ that allows TLR-7 agonists to induce SLE.