Search

This article is part of the Dendritic Cell Guidelines article series, which provides a collection of state-of-the-art protocols for the preparation, phenotype analysis by flow cytometry, generation, fluorescence microscopy, and functional characterization of mouse and human dendritic cells (DC) from lymphoid organs and various non-lymphoid tissues. This article provides protocols with top ticks and pitfalls for preparation and successful generation of mouse and human DC from different cellular sources, such as murine BM and HoxB8 cells, as well as human CD34 + cells from cord blood, BM, and peripheral blood or peripheral blood monocytes. We describe murine cDC1, cDC2, and pDC generation with Flt3L and the generation of BM-derived DC with GM-CSF. Protocols for human DC generation focus on CD34 + cell culture on OP9 cell layers for cDC1, cDC2, cDC3, and pDC subset generation and DC generation from peripheral blood monocytes (MoDC). Additional protocols include enrichment of murine DC subsets, CRISPR/Cas9 editing, and clinical grade human DC generation. While all protocols were written by experienced scientists who routinely use them in their work, this article was also peer-reviewed by leading experts and approved by all co-authors, making it an essential resource for basic and clinical DC immunologists., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

Aims: To elucidate the prognostic role of monocytes in the immune response of patients with coronary artery disease (CAD) at risk for life-threatening heart and lung injury as major complications of SARS-CoV-2 infection., Methods and Results: From February to April 2020, we prospectively studied a cohort of 96 participants comprising 47 consecutive patients with CAD and acute SARS-CoV-2 infection (CAD + SARS-CoV-2), 19 CAD patients without infections, and 30 healthy controls. Clinical assessment included blood sampling, echocardiography, and electrocardiography within 12 h of admission. Respiratory failure was stratified by the Horovitz Index (HI) as moderately/severely impaired when HI ≤200 mmHg. The clinical endpoint (EP) was defined as HI ≤200 mmHg with subsequent mechanical ventilation within a follow-up of 30 days. The numbers of CD14dimCD16+ non-classical monocytes in peripheral blood were remarkably low in CAD + SARS-CoV-2 compared with CAD patients without infection and healthy controls (P < 0.0001). Moreover, these CD14dimCD16 monocytes showed decreased expression of established markers of adhesion, migration, and T-cell activation (CD54, CD62L, CX3CR1, CD80, and HLA-DR). Decreased numbers of CD14dimCD16+ monocytes were associated with the occurrence of EP. Kaplan-Meier curves illustrate that CAD + SARS-CoV-2 patients with numbers below the median of CD14dimCD16+ monocytes (median 1443 cells/mL) reached EP significantly more often compared to patients with numbers above the median (log-rank 5.03, P = 0.025)., Conclusion: Decreased numbers of CD14dimCD16+ monocytes are associated with rapidly progressive respiratory failure in CAD + SARS-CoV-2 patients. Intensified risk assessments comprising monocyte sub- and phenotypes may help to identify patients at risk for respiratory failure., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.)

Sleep strongly impacts both humoral and cellular immunity; however, its acute effects on the innate immune defense against pathogens are unclear. Here, we elucidated in mice whether sleep affects the numbers and functions of innate immune cells and their defense against systemic bacterial infection. Sleep significantly increased numbers of classical monocytes in blood and spleen of mice that were allowed to sleep for six hours at the beginning of the normal resting phase compared to mice kept awake for the same time. The sleep-induced effect on classical monocytes was neither caused by alterations in corticosterone nor myelopoiesis, bone marrow egress or death of monocytes and did only partially involve Gαi-protein coupled receptors like chemokine receptor 2 (CCR2), but not the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) or lymphocyte function-associated antigen 1 (LFA-1). Notably, sleep suppressed the expression of the clock gene Arntl in splenic monocytes and the sleep-induced increase in circulating classical monocytes was abrogated in Arntl-deficient animals, indicating that sleep is a prerequisite for clock-gene driven rhythmic trafficking of classical monocytes. Sleep also enhanced the production of reactive oxygen species by monocytes and neutrophils. Moreover, sleep profoundly reduced bacterial load in blood and spleen of mice that were allowed to sleep before systemic bacterial infection and consequently increased survival upon infection. These data provide the first evidence that sleep enhances numbers and function of innate immune cells and therewith strengthens early defense against bacterial pathogens., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Staphylococcus aureus (Sa), as one of the major human pathogens, has very effective strategies to subvert the human immune system. Virulence of the emerging community-associated methicillin-resistant Sa (CA-MRSA) depends on the secretion of phenol-soluble modulin (PSM) peptide toxins e.g., by binding to and modulation of innate immune cells. Previously, by using mouse bone marrow-derived dendritic cells we demonstrated that PSMs in combination with various Toll-like receptor (TLR) ligands induce a tolerogenic DC phenotype (tDC) characterized by the production of IL-10 and impaired secretion of pro-inflammatory cytokines. Consequently, PSM-induced tDCs favored priming of CD4 + CD25 + FoxP3 + T regs with suppressor function while impairing the Th1 response. However, the relevance of these findings for the human system remained elusive. Here, we analyzed the impact of PSMα3 on the maturation, cytokine production, antigen uptake, and T cell stimulatory capacity of human monocyte-derived DCs (moDCs) treated simultaneously with either LPS (TLR4 ligand) or Sa cell lysate (TLR2 ligand). Herein, we demonstrate that PSMs indeed modulate human moDCs upon treatment with TLR2/4 ligands via multiple mechanisms, such as transient pore formation, impaired DC maturation, inhibited pro- and anti-inflammatory cytokine secretion, as well as reduced antigen uptake. As a result, the adaptive immune response was altered shown by an increased differentiation of naïve and even CD4 + T cells from patients with Th1/Th17-induced diseases (spondyloarthritis and rheumatoid arthritis) into CD4 + CD127 - CD25 hi CD45RA - FoxP3 hi regulatory T cells (T regs ) with suppressor function. This T reg induction was mediated most predominantly by direct DC-T-cell interaction. Thus, PSMs from highly virulent Sa strains affect DC functions not only in the mouse, but also in the human system, thereby modulating the adaptive immune response and probably increasing the tolerance toward the bacteria. Moreover, PSMα3 might be a novel peptide for tolerogenic DC induction that may be used for DC vaccination strategies.

The immune system protects from infections primarily by detecting and eliminating invading pathogens. Beside neutrophils, monocytes and dendritic cells (DCs) have been recently identified as important sentinels and effectors in combating microbial pathogens. In the steady state mononuclear phagocytes like monocytes and DCs patrol the blood and the tissues. Mammalian monocytes contribute to antimicrobial defense by supplying tissues with macrophage and DC precursors. DCs recognize pathogens and are essential in presenting antigens to initiate antigen-specific adaptive immune responses, thereby bridging the innate and adaptive immune systems. Both, monocytes and DCs play distinct roles in the shaping of immune response. In this review we will focus on the contributions of monocytes and lymphoid organ DCs to immune defense against microbial pathogens in the mouse and their dynamic regulation from steady state to infection., (Copyright © 2014 Elsevier GmbH. All rights reserved.)

Patients with cardiovascular disease (CVD) and acute SARS-CoV-2 infection might show an altered immune response during COVID-19. Twenty-three patients with CVD and SARS-CoV-2 infection were prospectively enrolled and received a cardiological assessment at study entry and during follow-up visit. Inclusion criteria of our study were age older than 18 years, presence of CVD, and acute SARS-CoV-2 infection. The median age of the patient cohort was 69 (IQR 55–79) years. 12 (52.2%) patients were men. Peripheral monocytes and chemokine/cytokine profiles were analysed. Numbers of classical and non-classical monocytes were significantly decreased during acute SARS-CoV-2 infection compared to 3-month recovery. While classical monocytes reached the expected level in peripheral blood after 3 months, the number of non-classical monocytes remained significantly reduced. All three monocyte subsets exhibited changes of established adhesion and activation markers. Interestingly, they also expressed higher levels of pro-inflammatory cytokines like macrophage migration inhibitory factor (MIF) at the time of recovery, although MIF was only slightly increased during the acute phase. Changes of monocyte phenotypes and increased MIF expression after 3-month recovery from acute SARS-CoV-2 infection may indicate persistent, possibly long-lasting, pro-inflammatory monocyte function in CVD patients. [ABSTRACT FROM AUTHOR]

• Microbial pathogens differently affect DC development either directly by PRRs and/or indirectly by inflammatory mediators. • Systemic bacterial infection results in impaired DC development but increased monocyte production. • Self-limiting infections promote innate immune memory and protect from secondary infections. • severe infections affect DC development leading to long-term immunosuppression. The immune system protects from infections primarily by detecting and eliminating invading pathogens. This is predominantly mediated by innate immune cells like neutrophils, monocytes and dendritic cells (DCs) expressing specific receptors recognizing pathogen-associated molecular patterns. DC activation by pathogens leads to the initiation of antigen-specific adaptive immune responses, thereby bridging the innate and adaptive immune systems. However, various pathogens have evolved immune evasion strategies to ensure their survival. In this review, we highlight recent findings on how various microorganisms or their structural features affect or modulate DC development and whether this has any consequences for a protective immune response. [ABSTRACT FROM AUTHOR]

Enteropathogenic Yersinia enterocolitica (Ye) enters the host via contaminated food. After colonisation of the small intestine Ye invades the Peyer’s patches (PPs) via M cells and disseminates to the mesenteric lymph nodes (MLNs), spleen and liver. Whether Ye uses other invasion routes and which pathogenicity factors are required remains elusive. Oral infection of lymphotoxin-β-receptor deficient mice lacking PPs and MLNs with Ye revealed similar bacterial load in the spleen 1 h post infection as wild-type mice, demonstrating a PP-independent dissemination route for Ye. Immunohistological analysis of the small intestine revealed Ye in close contact with mononuclear phagocytes (MPs), specifically CX 3 CR1 + monocyte-derived cells (MCs) as well as CD103 + dendritic cells (DCs). This finding was confirmed by flow cytometry and imaging flow cytometry analysis of lamina propria (LP) leukocytes showing CD103 + DCs and MCs with intracellular Ye. Uptake of Ye by LP CD103 + DCs and MCs was dependent on the pathogenicity factor invasin, whereas the adhesin YadA was dispensable as demonstrated by Ye deletion mutants. Furthermore, Ye were found exclusively associated with CD103 + DCs in the MLNs from wild-type mice, but not from CCR7 −/− mice, demonstrating a CCR7 dependent transport of Ye by CD103 + DCs from LP to the MLNs. In contrast, dissemination of Ye to the spleen was dependent on MCs as significantly less Ye could be recovered from the spleen of CX 3 CR1 GFP/GFP mice compared to wild-type mice. Altogether, MCs and CD103 + DCs contribute to immediate invasion and dissemination of Ye. This together with data from other bacteria suggests MPs as general pathogenic entry site in the intestine. [ABSTRACT FROM AUTHOR]

DCs are professional APCs playing a crucial role in the initiation of T-cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC-depletion in humans and mice. The mechanisms of pathogen-induced DC-depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC-development, one reason for DC-depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC-depletion and the impact of different bacteria on DC-development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram-positive and Gram-negative bacteria. This is associated with a TLR4- and IFN-γ−signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye-infection. Adoptive transfer experiments revealed that infection-induced monopoiesis occurs at the expense of DC-development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC-development leads to DC-depletion, possibly driving transient immunosuppression in bacterial sepsis. [ABSTRACT FROM AUTHOR]

Dendritic cells (DCs) are critical in host defense against infection. DC depletion is an early event in the course of sepsis that may impair the host defense mechanisms. Here, we addressed whether DC depletion and dysfunction are pathogen-independent, mediated via pattern recognition receptors, and are due to impaired DC development upon systemic infection with the Gram-negative bacterium Escherichia coli and the Gram-positive pathogen Staphylococcus aureus. Infection with E. coli and S. aureus led to reduced numbers of splenic DC subsets and of DC progenitors in the bone marrow (BM) with this effect persisting significantly longer in mice infected with S. aureus than with E. coli. The reduction of DC subsets and their progenitors was mainly TLR-independent as was the infection-induced monopoiesis. Moreover, de novo DC development was impaired in mice infected with S. aureus , and BM cells from E. coli or S. aureus infected mice favored macrophage differentiation in vitro. As a consequence of reduced DC numbers and their reduced expression of MHC II less CD4+ and CD8+ T cells, especially Th1 and IFN-γ producing CD8+ T cells, could be detected in S. aureus compared to E. coli infected mice. These differences are reflected in the rapid killing of E. coli as opposed to an increase in bacterial load in S. aureus. In summary, our study supports the idea that systemic bacterial infections generally affect the number and development of DCs and thereby the T cell responses, but the magnitude is pathogen-dependent. [ABSTRACT FROM AUTHOR]