HIV-1 Nef Protein Affects Cytokine and Extracellular Vesicles Production in the GEN2.2 Plasmacytoid Dendritic Cell Line

Plasmacytoid dendritic cells (pDCs) are a unique dendritic cell subset specialized in type I interferon production, whose role in Human Immunodeficiency Virus (HIV) infection and pathogenesis is complex and not yet well defined. Considering the crucial role of the accessory protein Nef in HIV pathogenicity, possible alterations in intracellular signalling and extracellular vesicle (EV) release induced by exogenous Nef on uninfected pDCs have been investigated. As an experimental model system, a human plasmacytoid dendritic cell line, GEN2.2, stimulated with a myristoylated recombinant NefSF2 protein was employed. In GEN2.2 cells, Nef treatment induced the tyrosine phosphorylation of STAT-1 and STAT-2 and the production of a set of cytokines, chemokines and growth factors including IP-10, MIP-1β, MCP-1, IL-8, TNF-α and G-CSF. The released factors differed both in type and amount from those released by macrophages treated with the same viral protein. Moreover, Nef treatment slightly reduces the production of small EVs, and the protein was found associated with the small (size < 200 nm) but not the medium/large vesicles (size > 200 nm) collected from GEN2.2 cells. These results add new information on the interactions between this virulence factor and uninfected pDCs, and may provide the basis for further studies on the interactions of Nef protein with primary pDCs.

Microbiome diversity declines while distinct expansions of Th17, iNKT, and dendritic cell subpopulations emerge after anastomosis surgery

Background Anastomotic failure causes morbidity and mortality even in technically correct anastomoses. Initial leaks must be prevented by mucosal reapproximation across the anastomosis. Healing is a concerted effort between intestinal epithelial cells (IECs), immune cells, and commensal bacteria. IEC TLR4 activation and signaling is required for mucosal healing, leading to inflammatory factor release that recruits immune cells to limit bacteria invasion. TLR4 absence leads to mucosal damage from loss in epithelial proliferation, attenuated inflammatory response, and bacteria translocation. We hypothesize after anastomosis, an imbalance in microbiota will occur due to a decrease in TLR4 expression and will lead to changes in the immune milieu. Results We isolated fecal content and small intestinal leukocytes from murine, Roux-en-Y and end-to-end anastomoses, to identify microbiome changes and subsequent alterations in the regulatory and pro-inflammatory immune cells 3 days post-operative. TLR4+ IECs were impaired after anastomosis. Microbiome diversity was reduced, with Firmicutes, Bacteroidetes, and Saccharibacteria decreased and Proteobacteria increased. A distinct TCRβhi CD4+ T cells subset after anastomosis was 10–20-fold greater than in control mice. 84% were Th17 IL-17A/F+ IL-22+ and/or TNFα+. iNKT cells were increased and TCRβhi. 75% were iNKT IL-10+ and 13% iNKTh17 IL-22+. Additionally, Treg IL-10+ and IL-22+ cells were increased. A novel dendritic cell subset was identified in anastomotic regions that was CD11bhi CD103mid and was 93% IL-10+. Conclusions This anastomotic study demonstrated a decrease in IEC TLR4 expression and microbiome diversity which then coincided with increased expansion of regulatory and pro-inflammatory immune cells and cytokines. Defining the anastomotic mucosal environment could help inform innovative therapeutics to target excessive pro-inflammatory invasion and microbiome imbalance.

Achieving dendritic cell subset-specific targeting in vivo by site-directed conjugation of targeting antibodies to nanocarriers

The major challenge of nanocarrier-based anti-cancer vaccination approaches is the targeted delivery of antigens and immunostimulatory agents to cells of interest, such as specific subtypes of dendritic cells (DCs), in order to induce robust antigen-specific anti-tumor responses. An undirected cell and body distribution of nanocarriers can lead to unwanted delivery to other immune cell types like macrophages reducing the vaccine efficacy. An often-used approach to overcome this issue is the surface functionalization of nanocarriers with targeting moieties, such as antibodies, mediating cell type-specific interaction. Numerous studies could successfully prove the targeting efficiency of antibody-conjugated carrier systems in vitro, however, most of them failed when targeting DCs in vivo that is partly due to cells of the reticuloendothelial system unspecifically clearing nanocarriers from the blood stream via Fc receptor ligation. Therefore, this study shows a surface functionalization strategy to site-specifically attach antibodies in an orientated direction onto the nanocarrier surface. Different DC-targeting antibodies, such as anti-CD11c, anti-CLEC9A, anti-DEC205 and anti-XCR1, were conjugated to the nanocarrier surface at their Fc domains. Anti-mouse CD11c antibody-conjugated nanocarriers specifically accumulated in the targeted organ (spleen) over time. Additionally, antibodies against CD11c and CLEC9A proved to specifically direct nanocarriers to the targeted DC subtype, conventional DCs type 1. In conclusion, site-directed antibody conjugation to nanocarriers is essential in order to avoid unspecific uptake by non-target cells while achieving antibody-specific targeting of DC subsets. This novel conjugation technique paves the way for the development of antibody-functionalized nanocarriers for DC-based vaccination approaches in the field of cancer immunotherapy.

Macrophage and dendritic cell subset composition can distinguish endotypes in adjuvant-induced asthma mouse models

Asthma is a heterogeneous disease with neutrophilic and eosinophilic asthma as the main endotypes that are distinguished according to the cells recruited to the airways and the related pathology. Eosinophilic asthma is the treatment-responsive endotype, which is mainly associated with allergic asthma. Neutrophilic asthma is a treatment-resistant endotype, affecting 5–10% of asthmatics. Although eosinophilic asthma is well-studied, a clear understanding of the endotypes is essential to devise effective diagnosis and treatment approaches for neutrophilic asthma. To this end, we directly compared adjuvant-induced mouse models of neutrophilic (CFA/OVA) and eosinophilic (Alum/OVA) asthma side-by-side. The immune response in the inflamed lung was analyzed by multi-parametric flow cytometry and immunofluorescence. We found that eosinophilic asthma was characterized by a preferential recruitment of interstitial macrophages and myeloid dendritic cells, whereas in neutrophilic asthma plasmacytoid dendritic cells, exudate macrophages, and GL7+ activated B cells predominated. This differential distribution of macrophage and dendritic cell subsets reveals important aspects of the pathophysiology of asthma and holds the promise to be used as biomarkers to diagnose asthma endotypes.

Macrophage and dendritic cell subset composition can distinguish endotypes in adjuvant-induced asthma mouse models

Asthma is a heterogeneous disease with neutrophilic and eosinophilic asthma as the main endotypes that are distinguished according to the cells recruited to the airways and the related pathology. Eosinophilic asthma is the treatment-responsive endotype, which is mainly associated with allergic asthma. Neutrophilic asthma is a treatment-resistant endotype, affecting 5-10% of asthmatics. Although eosinophilic asthma is well-studied, a clear understanding of the endotypes is essential to devise effective diagnosis and treatment approaches for neutrophilic asthma. To this end, we directly compared adjuvant-induced mouse models of neutrophilic (CFA/OVA) and eosinophilic (Alum/OVA) asthma side-by-side. The immune response in the inflamed lung was analyzed by multi-parametric flow cytometry and immunofluorescence. We found that eosinophilic asthma was characterized by a preferential recruitment of interstitial macrophages and myeloid dendritic cells, whereas in neutrophilic asthma plasmacytoid dendritic cells, exudate macrophages, and GL7 + activated B cells predominated. This differential distribution of macrophage and dendritic cell subsets reveals important aspects of the pathophysiology of asthma and holds the promise to be used as biomarkers to diagnose asthma endotypes.

Development of an Inflammatory CD14+ Dendritic Cell Subset in Humanized Mice

Humanized mouse models are attractive experimental models for analyzing the development and functions of human dendritic cells (DCs) in vivo. Although various types of DC subsets, including DC type 3 (DC3s), have been identified in humans, it remains unclear whether humanized mice can reproduce heterogeneous DC subsets. CD14, classically known as a monocyte/macrophage marker, is reported as an indicator of DC3s. We previously observed that some CD14+ myeloid cells expressed CD1c, a pan marker for bona fide conventional DC2 (cDC2s), in humanized mouse models in which human FLT3L and GM-CSF genes were transiently expressed using in vivo transfection (IVT). Here, we aimed to elucidate the identity of CD14+CD1c+ DC-like cells in humanized mouse models. We found that CD14+CD1c+ cells were phenotypically different from cDC2s; CD14+CD1c+ cells expressed CD163 but not CD5, whereas cDC2s expressed CD5 but not CD163. Furthermore, CD14+CD1c+ cells primed and polarized naïve CD4+ T cells toward IFN-γ+ Th1 cells more profoundly than cDC2s. Transcriptional analysis revealed that CD14+CD1c+ cells expressed several DC3-specific transcripts, such as CD163, S100A8, and S100A9, and were clearly segregated from cDC2s and monocytes. When lipopolysaccharide was administered to the humanized mice, the frequency of CD14+CD1c+ cells producing IL-6 and TNF-α was elevated, indicating a pro-inflammatory signature. Thus, humanized mice are able to sustain development of functional CD14+CD1c+ DCs, which are equivalent to DC3 subset observed in humans, and they could be useful for analyzing the development and function of DC3s in vivo.

Human CD141+ dendritic cells (cDC1) are impaired in patients with advanced melanoma but can be targeted to enhance anti-PD-1 in a humanized mouse model

BackgroundThe conventional type 1 dendritic cell subset (cDC1) is indispensable for tumor immune responses and the efficacy of immune checkpoint inhibitor (ICI) therapies in animal models but little is known about the role of the human CD141+ DC cDC1 equivalent in patients with melanoma.MethodsWe developed a flow cytometry assay to quantify and characterize human blood DC subsets in healthy donors and patients with stage 3 and stage 4 metastatic melanoma. To examine whether harnessing CD141+ DCs could improve responses to ICIs in human melanoma, we developed a humanized mouse model by engrafting immunodeficient NSG-SGM3 mice with human CD34+ hematopoietic stem cells (HSCs) from umbilical cord blood followed by transplantation of a human melanoma cell line and treatment with anti-programmed cell death protein-1 (anti-PD-1).ResultsBlood CD141+ DC numbers were significantly reduced in patients with stage 4 melanoma compared with healthy controls. Moreover, CD141+ DCs in patients with melanoma were selectively impaired in their ability to upregulate CD83 expression after stimulation with toll-like receptor 3 (TLR3) and TLR7/8 agonists ex vivo. Although DC numbers did not correlate with responses to anti-PD-1 and/or anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) ICIs, their numbers and capacity to upregulate CD83 declined further during treatment in non-responding patients. Treatment with anti-PD-1 was ineffective at controlling tumor growth in humanized mice but efficacy was enhanced by indirectly expanding and activating DCs in vivo with fms-like tyrosine kinase-3 ligand (Flt3L) and a TLR3 agonist. Moreover, intratumoral injections of CD141+ DCs resulted in reduced tumor growth when combined with anti-PD-1 treatment.ConclusionsThese data illustrate quantitative and qualitative impairments in circulating CD141+ DCs in patients with advanced melanoma and that increasing CD141+ DC number and function is an attractive strategy to enhance immunogenicity and response rates to ICIs.

Dendritic cell responses to Plasmodium falciparum in a malaria-endemic setting

Background Plasmodium falciparum causes the majority of malaria cases worldwide and children in sub-Saharan Africa are the most vulnerable group affected. Non-sterile clinical immunity that protects from symptoms develops slowly and is relatively short-lived. Moreover, current malaria vaccine candidates fail to induce durable high-level protection in endemic settings, possibly due to the immunomodulatory effects of the malaria parasite itself. Because dendritic cells play a crucial role in initiating immune responses, the aim of this study was to better understand the impact of cumulative malaria exposure as well as concurrent P. falciparum infection on dendritic cell phenotype and function. Methods In this cross-sectional study, the phenotype and function of dendritic cells freshly isolated from peripheral blood samples of Malian adults with a lifelong history of malaria exposure who were either uninfected (n = 27) or asymptomatically infected with P. falciparum (n = 8) was assessed. Additionally, plasma cytokine and chemokine levels were measured in these adults and in Malian children (n = 19) with acute symptomatic malaria. Results With the exception of lower plasmacytoid dendritic cell frequencies in asymptomatically infected Malian adults, peripheral blood dendritic cell subset frequencies and HLA-DR surface expression did not differ by infection status. Peripheral blood myeloid dendritic cells of uninfected Malian adults responded to in vitro stimulation with P. falciparum blood-stage parasites by up-regulating the costimulatory molecules HLA-DR, CD80, CD86 and CD40 and secreting IL-10, CXCL9 and CXCL10. In contrast, myeloid dendritic cells of asymptomatically infected Malian adults exhibited no significant responses above the uninfected red blood cell control. IL-10 and CXCL9 plasma levels were elevated in both asymptomatic adults and children with acute malaria. Conclusions The findings of this study indicate that myeloid dendritic cells of uninfected adults with a lifelong history of malaria exposure are able to up-regulate co-stimulatory molecules and produce cytokines. Whether mDCs of malaria-exposed individuals are efficient antigen-presenting cells capable of mounting an appropriate immune response remains to be determined. The data also highlights IL-10 and CXCL9 as important factors in both asymptomatic and acute malaria and add to the understanding of asymptomatic P. falciparum infections in malaria-endemic areas.