Recovery of systemic hyperinflammation in patients with severe SARS-CoV-2 infection.

INTRODUCTION: Patients with cardiovascular disease (CVD) and acute SARS-CoV-2 infection might show an altered immune response during COVID-19. MATERIAL AND METHODS: 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. RESULTS: 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. DISCUSSION: 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. CONCLUSION: 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.

Dysfunctional dendritic cells limit antigen-specific T cell response in glioma.

BACKGROUND: Dendritic cells (DC), the most potent professional antigen presenting cells capable of effective cross-presentation, have been demonstrated to license T helper cells to induce antitumor immunity in solid tumors. Specific DC subtypes are recruited to the injured brain by microglial chemokines, locally adapting to distinct transcriptional profiles. In isocitrate dehydrogenase (IDH) type 1 mutant gliomas, monocyte-derived macrophages have recently been shown to display an attenuated intratumoral antigen presentation capacity as consequence of the local accumulation of the oncometabolite R-2-hydroxyglutarate. The functionality and the contribution of DC to the IDH-mutant tumor microenvironment (TME) remains unclear. METHODS: Frequencies and intratumoral phenotypes of human DC in IDH-wildtype (IDHwt) and -mutant high-grade gliomas are comparatively assessed by transcriptomic and proteomic profiling. DC functionality is investigated in experimental murine glioblastomas expressing the model antigen ovalbumin. Single-cell sequencing-based pseudotime analyses and spectral flow cytometric analyses are used to profile DC states longitudinally. RESULTS: DC are present in primary and recurrent high-grade gliomas and interact with other immune cell types within the TME. In murine glioblastomas, we find an IDH-status-associated major histocompatibility class I-restricted cross-presentation of tumor antigens by DC specifically in the tumor but not in meninges or secondary lymphoid organs of tumor-bearing animals. In single-cell sequencing-based pseudotime and longitudinal spectral flow cytometric analyses, we demonstrate an IDH-status-dependent differential, exclusively microenvironmental education of DC. CONCLUSIONS: Glioma-associated DCs are relevantly abundant in human IDHwt and mutant tumors. Glioma IDH mutations result in specifically educated, dysfunctional DCs via paracrine reprogramming of infiltrating monocytes, providing the basis for combinatorial immunotherapy concepts against IDH mutant gliomas.

ACKR3 regulates platelet activation and ischemia-reperfusion tissue injury.

Platelet activation plays a critical role in thrombosis. Inhibition of platelet activation is a cornerstone in treatment of acute organ ischemia. Platelet ACKR3 surface expression is independently associated with all-cause mortality in CAD patients. In a novel genetic mouse strain, we show that megakaryocyte/platelet-specific deletion of ACKR3 results in enhanced platelet activation and thrombosis in vitro and in vivo. Further, we performed ischemia/reperfusion experiments (transient LAD-ligation and tMCAO) in mice to assess the impact of genetic ACKR3 deficiency in platelets on tissue injury in ischemic myocardium and brain. Loss of platelet ACKR3 enhances tissue injury in ischemic myocardium and brain and aggravates tissue inflammation. Activation of platelet-ACKR3 via specific ACKR3 agonists inhibits platelet activation and thrombus formation and attenuates tissue injury in ischemic myocardium and brain. Here we demonstrate that ACKR3 is a critical regulator of platelet activation, thrombus formation and organ injury following ischemia/reperfusion.

Systemic bacterial infections affect dendritic cell development and function.

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.

Platelet Activation and Plasma Levels of Furin Are Associated With Prognosis of Patients With Coronary Artery Disease and COVID-19.

[Figure: see text].

Numbers and phenotype of non-classical CD14dimCD16+ monocytes are predictors of adverse clinical outcome in patients with coronary artery disease and severe SARS-CoV-2 infection.

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.

Sleep enhances numbers and function of monocytes and improves bacterial infection outcome in mice.

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.

PSM Peptides From Community-Associated Methicillin-Resistant Staphylococcus aureus Impair the Adaptive Immune Response via Modulation of Dendritic Cell Subsets in vivo.

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factors phenol-soluble-modulins (PSMs) produced by community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) strains induce tolerogenic DCs upon Toll-like receptor activation via the p38-CREB-IL-10 pathway in vitro. Here, we addressed the hypothesis that S. aureus PSMs disturb the adaptive immune response via modulation of DC subsets in vivo. Using a systemic mouse infection model we found that S. aureus reduced the numbers of splenic DC subsets, mainly CD4+ and CD8+ DCs independently of PSM secretion. S. aureus infection induced upregulation of the C-C motif chemokine receptor 7 (CCR7) on the surface of all DC subsets, on CD4+ DCs in a PSM-dependent manner, together with increased expression of MHCII, CD86, CD80, CD40, and the co-inhibitory molecule PD-L2, with only minor effects of PSMs. Moreover, PSMs increased IL-10 production in the spleen and impaired TNF production by CD4+ DCs. Besides, S. aureus PSMs reduced the number of CD4+ T cells in the spleen, whereas CD4+CD25+Foxp3+ regulatory T cells (Tregs) were increased. In contrast, Th1 and Th17 priming and IFN-γ production by CD8+ T cells were impaired by S. aureus PSMs. Thus, PSMs from highly virulent S. aureus strains modulate the adaptive immune response in the direction of tolerance by affecting DC functions.

Staphylococcus aureus PSM Peptides Modulate Human Monocyte-Derived Dendritic Cells to Prime Regulatory T Cells.

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+ Tregs 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-CD25hiCD45RA-FoxP3hi regulatory T cells (Tregs) with suppressor function. This Treg 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.

Staphylococcus aureus PSM peptides induce tolerogenic dendritic cells upon treatment with ligands of extracellular and intracellular TLRs.

Dendritic cells (DCs) are key players of the immune system and thus a target for immune evasion by pathogens. We recently showed that the virulence factor phenol-soluble modulin (PSM) produced by community-associated methicillin-resistant Staphylococcus aureus strains induces tolerogenic DCs upon Toll-like receptor (TLR) 2 activation via the p38-CREB-IL-10 pathway. Here, we addressed the question whether this tolerogenic phenotype of DCs induced by PSMs is specific for TLR2 activation. Therefore, bone marrow-derived DCs were treated with various ligands for extracellular and intracellular TLRs simultaneously with PSMα3. We show that PSMα3 modulates antigen uptake, maturation and cytokine production of DCs activated by TLR1/2, TLR2/6, TLR4, TLR7, and TLR9. Pre-incubation of DCs with a p38 MAP kinase inhibitor prevented the PSMα3-induced IL-10 secretion, as well as MHC class II up-regulation upon TLR activation. In consequence, the tolerogenic DCs induced by PSMα3 in response to several TLR ligands promoted priming of regulatory T cells. Thus, PSMs could be useful as inducers of tolerogenic DCs upon TLR ligand stimulation for therapeutic applications.

Mononuclear phagocytes contribute to intestinal invasion and dissemination of Yersinia enterocolitica.

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-ß-receptor deficient mice lacking PPs and MLNs with Ye revealed similar bacterial load in the spleen 1h 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 CX3CR1(+) 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 CX3CR1(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.

PSM Peptides of Staphylococcus aureus Activate the p38-CREB Pathway in Dendritic Cells, Thereby Modulating Cytokine Production and T Cell Priming.

The challenging human pathogen Staphylococcus aureus has highly efficient immune evasion strategies for causing a wide range of diseases, from skin and soft tissue to life-threatening infections. Phenol-soluble modulin (PSM) peptides are major pathogenicity factors of community-associated methicillin-resistant S. aureus strains. In previous work, we demonstrated that PSMs in combination with TLR2 ligand from S. aureus induce tolerogenic dendritic cells (DCs) characterized by the production of high amounts of IL-10, but no proinflammatory cytokines. This in turn promotes the activation of regulatory T cells while impairing Th1 response; however, the signaling pathways modulated by PSMs remain elusive. In this study, we analyzed the effects of PSMs on signaling pathway modulation downstream of TLR2. TLR2 stimulation in combination with PSMα3 led to increased and prolonged phosphorylation of NF-κB, ERK, p38, and CREB in mouse bone marrow-derived DCs compared with single TLR2 activation. Furthermore, inhibition of p38 and downstream MSK1 prevented IL-10 production, which in turn reduced the capacity of DCs to activate regulatory T cells. Interestingly, the modulation of the signaling pathways by PSMs was independent of the known receptor for PSMs, as shown by experiments with DCs lacking the formyl peptide receptor 2. Instead, PSMs penetrate the cell membrane most likely by transient pore formation. Moreover, colocalization of PSMs and p38 was observed near the plasma membrane in the cytosol, indicating a direct interaction. Thus, PSMs from S. aureus directly modulate the signaling pathway p38-CREB in DCs, thereby impairing cytokine production and in consequence T cell priming to increase the tolerance toward the pathogen.

Innate immune system favors emergency monopoiesis at the expense of DC-differentiation to control systemic bacterial infection in mice.

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.

Depletion of dendritic cells enhances innate anti-bacterial host defense through modulation of phagocyte homeostasis.

Dendritic cells (DCs) as professional antigen-presenting cells play an important role in the initiation and modulation of the adaptive immune response. However, their role in the innate immune response against bacterial infections is not completely defined. Here we have analyzed the role of DCs and their impact on the innate anti-bacterial host defense in an experimental infection model of Yersinia enterocolitica (Ye). We used CD11c-diphtheria toxin (DT) mice to deplete DCs prior to severe infection with Ye. DC depletion significantly increased animal survival after Ye infection. The bacterial load in the spleen of DC-depleted mice was significantly lower than that of control mice throughout the infection. DC depletion was accompanied by an increase in the serum levels of CXCL1, G-CSF, IL-1α, and CCL2 and an increase in the numbers of splenic phagocytes. Functionally, splenocytes from DC-depleted mice exhibited an increased bacterial killing capacity compared to splenocytes from control mice. Cellular studies further showed that this was due to an increased production of reactive oxygen species (ROS) by neutrophils. Adoptive transfer of neutrophils from DC-depleted mice into control mice prior to Ye infection reduced the bacterial load to the level of Ye-infected DC-depleted mice, suggesting that the increased number of phagocytes with additional ROS production account for the decreased bacterial load. Furthermore, after incubation with serum from DC-depleted mice splenocytes from control mice increased their bacterial killing capacity, most likely due to enhanced ROS production by neutrophils, indicating that serum factors from DC-depleted mice account for this effect. In summary, we could show that DC depletion triggers phagocyte accumulation in the spleen and enhances their anti-bacterial killing capacity upon bacterial infection.