Cytomegalovirus subverts macrophage identity.

Cytomegaloviruses (CMVs) have co-evolved with their mammalian hosts for millions of years, leading to remarkable host specificity and high infection prevalence. Macrophages, which already populate barrier tissues in the embryo, are the predominant immune cells at potential CMV entry sites. Here we show that, upon CMV infection, macrophages undergo a morphological, immunophenotypic, and metabolic transformation process with features of stemness, altered migration, enhanced invasiveness, and provision of the cell cycle machinery for viral proliferation. This complex process depends on Wnt signaling and the transcription factor ZEB1. In pulmonary infection, mouse CMV primarily targets and reprograms alveolar macrophages, which alters lung physiology and facilitates primary CMV and secondary bacterial infection by attenuating the inflammatory response. Thus, CMV profoundly perturbs macrophage identity beyond established limits of plasticity and rewires specific differentiation processes, allowing viral spread and impairing innate tissue immunity.

DNA Damage Signaling Instructs Polyploid Macrophage Fate in Granulomas.

Granulomas are immune cell aggregates formed in response to persistent inflammatory stimuli. Granuloma macrophage subsets are diverse and carry varying copy numbers of their genomic information. The molecular programs that control the differentiation of such macrophage populations in response to a chronic stimulus, though critical for disease outcome, have not been defined. Here, we delineate a macrophage differentiation pathway by which a persistent Toll-like receptor (TLR) 2 signal instructs polyploid macrophage fate by inducing replication stress and activating the DNA damage response. Polyploid granuloma-resident macrophages formed via modified cell divisions and mitotic defects and not, as previously thought, by cell-to-cell fusion. TLR2 signaling promoted macrophage polyploidy and suppressed genomic instability by regulating Myc and ATR. We propose that, in the presence of persistent inflammatory stimuli, pathways previously linked to oncogene-initiated carcinogenesis instruct a long-lived granuloma-resident macrophage differentiation program that regulates granulomatous tissue remodeling.

A Subset of Skin Macrophages Contributes to the Surveillance and Regeneration of Local Nerves.

The skin comprises tissue macrophages as the most abundant resident immune cell type. Their diverse tasks including resistance against invading pathogens, attraction of bypassing immune cells from vessels, and tissue repair require dynamic specification. Here, we delineated the postnatal development of dermal macrophages and their differentiation into subsets by adapting single-cell transcriptomics, fate mapping, and imaging. Thereby we identified a phenotypically and transcriptionally distinct subset of prenatally seeded dermal macrophages that self-maintained with very low postnatal exchange by hematopoietic stem cells. These macrophages specifically interacted with sensory nerves and surveilled and trimmed the myelin sheath. Overall, resident dermal macrophages contributed to axon sprouting after mechanical injury. In summary, our data show long-lasting functional specification of macrophages in the dermis that is driven by stepwise adaptation to guiding structures and ensures codevelopment of ontogenetically distinct cells within the same compartment.

Helminthic dehydrogenase drives PGE2 and IL-10 production in monocytes to potentiate Treg induction.

Immunoregulation of inflammatory, infection-triggered processes in the brain constitutes a central mechanism to control devastating disease manifestations such as epilepsy. Observational studies implicate the viability of Taenia solium cysts as key factor determining severity of neurocysticercosis (NCC), the most common cause of epilepsy, especially in children, in Sub-Saharan Africa. Viable, in contrast to decaying, cysts mostly remain clinically silent by yet unknown mechanisms, potentially involving Tregs in controlling inflammation. Here, we show that glutamate dehydrogenase from viable cysts instructs tolerogenic monocytes to release IL-10 and the lipid mediator PGE2 . These act in concert, converting naive CD4+ T cells into CD127- CD25hi FoxP3+ CTLA-4+ Tregs, through the G protein-coupled receptors EP2 and EP4 and the IL-10 receptor. Moreover, while viable cyst products strongly upregulate IL-10 and PGE2 transcription in microglia, intravesicular fluid, released during cyst decay, induces pro-inflammatory microglia and TGF-ß as potential drivers of epilepsy. Inhibition of PGE2 synthesis and IL-10 signaling prevents Treg induction by viable cyst products. Harnessing the PGE2 -IL-10 axis and targeting TGF-ß signaling may offer an important therapeutic strategy in inflammatory epilepsy and NCC.

Paradoxical immunodeficiencies-When failures of innate immunity cause immunopathology.

Innate immunity facilitates immediate defense against invading pathogens throughout all organs and tissues but also mediates tissue homeostasis and repair, thereby playing a key role in health and development. Recognition of pathogens is mediated by germline-encoded PRRs. Depending on the specific PRRs triggered, ligand binding leads to phagocytosis and pathogen killing and the controlled release of immune-modulatory factors such as IFNs, cytokines, or chemokines. PRR-mediated and other innate immune responses do not only prevent uncontrolled replication of intruding pathogens but also contribute to the tailoring of an effective adaptive immune response. Therefore, hereditary or acquired immunodeficiencies impairing innate responses may paradoxically cause severe immunopathology in patients. This can occur in the context of, but also independently of an increased microbial burden. It can include pathogen-dependent organ damage, autoinflammatory syndromes, and neurodevelopmental or neurodegenerative diseases. Here, we discuss the current state of research of several different such immune paradoxes. Understanding the underlying mechanisms causing immunopathology as a consequence of failures of innate immunity may help to prevent life-threatening disease.

Origin and Differentiation of Nerve-Associated Macrophages.

The mature peripheral nervous system is a steady network structure yet shows remarkable regenerative properties. The interaction of peripheral nerves with myeloid cells has largely been investigated in the context of damage, following trauma or infection. Recently, specific macrophages dedicated to homeostatic peripheral nerves have come into focus. These macrophages are defined by tissue and nerve type, are seeded in part prenatally, and self-maintain via proliferation. Thus, they are markedly distinct from monocyte-derived macrophages invading after local disturbance of nerve integrity. The phenotypic and transcriptional adaptation of macrophages to the discrete nervous niche may exert axon guidance and nerve regeneration and thus contribute to the stability of the peripheral nervous network. Deciphering these conserved macrophage-nerve interactions offers new translational perspectives for chronic diseases of the peripheral nervous system, such as diabetic neuropathy and pain.

Transmission of Severe Acute Respiratory Syndrome Coronavirus 2 in Households with Children, Southwest Germany, May-August 2020.

Resolving the role of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission in households with members from different generations is crucial for containing the current pandemic. We conducted a large-scale, multicenter, cross-sectional seroepidemiologic household transmission study in southwest Germany during May 11-August 1, 2020. We included 1,625 study participants from 405 households that each had ≥1 child and 1 reverse transcription PCR-confirmed SARS-CoV-2-infected index case-patient. The overall secondary attack rate was 31.6% and was significantly higher in exposed adults (37.5%) than in children (24.6%-29.2%; p = <0.015); the rate was also significantly higher when the index case-patient was >60 years of age (72.9%; p = 0.039). Other risk factors for infectiousness of the index case-patient were SARS-CoV-2-seropositivity (odds ratio [OR] 27.8, 95% CI 8.26-93.5), fever (OR 1.93, 95% CI 1.14-3.31), and cough (OR 2.07, 95% CI 1.21-3.53). Secondary infections in household contacts generate a substantial disease burden.

High diagnostic yield of endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) in the diagnosis of adolescent pulmonary tuberculosis.

BACKGROUND: The microbiological diagnosis of pulmonary tuberculosis (Tb) in a pediatric population is hampered by both low pathogen burden and noncompliance with sputum sampling. Although endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) has been found useful for the evaluation of mediastinal pathologies in adults, for children, sparse data are available. Here, we have evaluated EBUS-TBNA as a diagnostic procedure in children and adolescents with suspected pulmonary Tb. METHODS: In this retrospective analysis, we reviewed the charts of unaccompanied refugee minors (URM) who were admitted between January 2016 and July 2018 and who, during their initial medical screening upon arrival in Germany, were found to have abnormal radiological pulmonary and mediastinal findings and/or immunological results indicative of Tb. For each patient, basic sociodemographic data, clinical features and data on diagnostic procedures performed were assessed. These included imaging, immunodiagnostic tests and microbiological data derived from sputum, bronchoalveolar lavage, EBUS-TBNA, bronchoscopy and pleural fluid sampling. All patients who underwent invasive sampling procedures were included in the study. RESULTS: Out of 42 URM with suspected Tb, 34 fulfilled the study's inclusion criteria. Ages ranged from 14 to 17 years. All were of African origin, with 70.0% coming from Somalia, Eritrea and Ethiopia. Among the 21 patients for whom EBUS-TBNA was performed, the diagnostic yield was high: 66.7% positive results (MTb detected either by acid-fast stain, culture or PCR in 4.8, 42.9 and 61.9% of samples, respectively). Multidrug-resistant MTb was found in two patients from Somalia. No complications were associated with the procedure. Overall, pulmonary Tb was diagnosed in 29 patients (85.3%), miliary Tb in two patients (5.9%) and latent Tb in three patients (8.8%). CONCLUSIONS: EBUS-TBNA is a sensitive and safe method with high diagnostic yield in the evaluation of pediatric patients with mediastinal pathology and suspected Tb.

Macrophages Are a Potent Source of Streptococcus-Induced IFN-ß.

IFN-ß essentially modulates the host response against mucocutaneous colonizers and potential pathogens, such as group B Streptococcus (GBS). It has been reported that the dominant signaling cascade driving IFN-ß in macrophages (MΦ) in streptococcal infection is the cGAS-STING pathway, whereas conventional dendritic cells (DC) exploit endosomal recognition by intracellular TLRs. In this study, we revisited this issue by precisely monitoring the phenotypic dynamics in mixed mouse MΦ/DC cultures with GM-CSF, which requires snapshot definition of cellular identities. We identified four mononuclear phagocyte populations, of which two were transcriptionally and morphologically distinct MΦ-DC-like subsets, and two were transitional types. Notably, GBS induced a TLR7-dependent IFN-ß signal only in MΦ-like but not in DC-like cells. IFN-ß induction did not require live bacteria (i.e., the formation of cytolytic toxins), which are essential for IFN-ß induction via cGAS-STING. In contrast to IFN-ß, GBS induced TNF-α independently of TLR7. Subsequent to the interaction with streptococci, MΦ changed their immunophenotype and gained some typical DC markers and DC-like morphology. In summary, we identify IFN-ß formation as part of the antistreptococcal repertoire of GM-CSF differentiated MΦ in vitro and in vivo and delineate their plasticity.

Risk Factors for Complicated Lymphadenitis Caused by Nontuberculous Mycobacteria in Children.

Nontuberculous mycobacteria (NTM) are an emerging cause of infections, including chronic lymphadenitis in children. To identify risk factors for NTM lymphadenitis, particularly complicated disease, we collected epidemiologic, clinical, and microbiological data on 138 cases of NTM lymphadenitis in children across 13 centers in Germany and Austria. We assessed lifestyle factors but did not identify specific risk behaviors. We noted that more cases of NTM lymphadenitis occurred during cold months than during warm months. Moreover, we noted female sex and age <5.5 years as potential risk factors. Complete extirpation of the affected lymph node appeared to be the best therapeutic measure. We integrated the study data to develop a simple risk score to predict unfavorable clinical outcomes for NTM lymphadenitis.

Comprehensive infectious disease screening in a cohort of unaccompanied refugee minors in Germany from 2016 to 2017: A cross-sectional study.

BACKGROUND: Information regarding the prevalence of infectious diseases (IDs) in child and adolescent refugees in Europe is scarce. Here, we evaluate a standardized ID screening protocol in a cohort of unaccompanied refugee minors (URMs) in a municipal region of southwest Germany. METHODS AND FINDINGS: From January 2016 to December 2017, we employed a structured questionnaire to screen a cohort of 890 URMs. Collecting sociodemographic information and medical history, we also performed a standardized diagnostics panel, including complete blood count, urine status, microbial stool testing, tuberculosis (TB) screening, and serologies for hepatitis B virus (HBV) and human immunodeficiency virus (HIV). The mean age was 16.2 years; 94.0% were male, and 93.6% originated from an African country. The most common health complaints were dental problems (66.0%). The single most frequent ID was scabies (14.2%). Of the 776 URMs originating from high-prevalence countries, 7.7% and 0.4% tested positive for HBV and HIV, respectively. Nineteen pathogens were detected in a total of 119 stool samples (16.0% positivity), with intestinal schistosomiasis being the most frequent pathogen (6.7%). Blood eosinophilia proved to be a nonspecific criterion for the detection of parasitic infections. Active pulmonary TB was identified in 1.7% of URMs screened. Of note, clinical warning symptoms (fever, cough >2 weeks, and weight loss) were insensitive parameters for the identification of patients with active TB. Study limitations include the possibility of an incomplete eosinophilia workup (as no parasite serologies or malaria diagnostics were performed), as well as the inherent selection bias in our cohort because refugee populations differ across Europe. CONCLUSIONS: Our study found that standardized ID screening in a URM cohort was practicable and helped collection of relevant patient data in a thorough and time-effective manner. However, screening practices need to be ameliorated, especially in relation to testing for parasitic infections. Most importantly, we found that only a minority of infections were able to be detected clinically. This underscores the importance of active surveillance of IDs among refugees.

Eosinophilia and reduced STAT3 signaling affect neutrophil cell death in autosomal-dominant Hyper-IgE syndrome.

The autosomal-dominant hyper-IgE syndrome (HIES), caused by mutations in STAT3, is a rare primary immunodeficiency that predisposes to mucocutaneous candidiasis and staphylococcal skin and lung infections. This infection phenotype is suggestive of defects in neutrophils, but data on neutrophil functions in HIES are inconsistent. This study was undertaken to functionally characterize neutrophils in STAT3-deficient HIES patients and to analyze whether the patients` eosinophilia affects the neutrophil phenotype in S. aureus infection. Neutrophil functions and cell death kinetics were studied in eight STAT3-deficient patients. Moreover, the response of STAT3-deficient neutrophils to S. aureus and the impact of autologous eosinophils on pathogen-induced cell death were analyzed. No specific aberrations in neutrophil functions were detected within this cohort. However, the half-life of STAT3-deficient neutrophils ex vivo was reduced, which was partially attributable to the presence of eosinophils. Increased S. aureus-induced cell lysis, dependent on the staphylococcal virulence controlling accessory gene regulator (agr)-locus, was observed in STAT3-deficient neutrophils and upon addition of eosinophils. Accelerated neutrophil cell death kinetics may underlie the reported variability in neutrophil function testing in HIES. Increased S. aureus-induced lysis of STAT3-deficient neutrophils might affect pathogen control and contribute to tissue destruction during staphylococcal infections in HIES.

Mycobacteria exploit nitric oxide-induced transformation of macrophages into permissive giant cells.

Immunity to mycobacteria involves the formation of granulomas, characterized by a unique macrophage (MΦ) species, so-called multinucleated giant cells (MGC). It remains unresolved whether MGC are beneficial to the host, that is, by prevention of bacterial spread, or whether they promote mycobacterial persistence. Here, we show that the prototypical antimycobacterial molecule nitric oxide (NO), which is produced by MGC in excessive amounts, is a double-edged sword. Next to its antibacterial capacity, NO propagates the transformation of MΦ into MGC, which are relatively permissive for mycobacterial persistence. The mechanism underlying MGC formation involves NO-induced DNA damage and impairment of p53 function. Moreover, MGC have an unsurpassed potential to engulf mycobacteria-infected apoptotic cells, which adds a further burden to their antimycobacterial capacity. Accordingly, mycobacteria take paradoxical advantage of antimicrobial cellular efforts by driving effector MΦ into a permissive MGC state.

A DELPHI study on aspects of study design to overcome knowledge gaps on the burden of disease caused by serogroup B invasive meningococcal disease.

BACKGROUND: Value assessment of vaccination programs against serogroup B invasive meningococcal disease (IMD) is on the agenda of public health authorities. Current evidence on the burden due to IMD is unfit for pinning down the nature and magnitude of the full social and economic costs of IMD for two reasons. First, the concepts and components that need to be studied are not agreed, and second, measures of the concepts that have been studied are weak and inconsistent. Thus, the economic evaluation of the available serogroup B meningococcal (MenB) vaccines is difficult. The aims of this DELPHI study are to: (1) agree on the concepts and components determining the burden of MenB diseases that need to be studied; and (2) seek consensus on appropriate methods and study designs to measure quality of life (QoL) associated with MenB induced long-term sequelae in future studies. METHODS: We designed a DELPHI questionnaire based on the findings of a recent systematic review on the QoL associated with IMD-induced long-term sequelae, and iteratively interviewed a panel of international experts, including physicians, health economists, and patient representatives. Experts were provided with a controlled feedback based on the results of the previous round. RESULTS: Experts reached consensus on all questions after two DELPHI rounds. Major gaps in the literature relate (i) to the classification of sequelae, which allows differentiation of severity levels, (ii) to the choice of QoL measures, and (iii) to appropriate data sources to examine long-term changes and deficits in patients' QoL. CONCLUSIONS: Better conceptualisation of the structure of IMD-specific sequelae and of how their diverse forms of severity might impact the QoL of survivors of IMD as well as their family network and care-providers is needed to generate relevant, reliable and generalisable data on QoL in the future. The results of this DELPHI panel provide useful guidance on how to choose the study design, target population and appropriate QoL measures for future research and hence, help promote the appropriateness and consistency in study methodology and sample characteristics.

Streptococci Engage TLR13 on Myeloid Cells in a Site-Specific Fashion.

Streptococci are common human colonizers with a species-specific mucocutaneous distribution. At the same time, they are among the most important and most virulent invasive bacterial pathogens. Thus, site-specific cellular innate immunity, which is predominantly executed by resident and invading myeloid cells, has to be adapted with respect to streptococcal sensing, handling, and response. In this article, we show that TLR13 is the critical mouse macrophage (MΦ) receptor in the response to group B Streptococcus, both in bone marrow-derived MΦs and in mature tissue MΦs, such as those residing in the lamina propria of the colon and the dermis, as well as in microglia. In contrast, TLR13 and its chaperone UNC-93B are dispensable for a potent cytokine response of blood monocytes to group B Streptococcus, although monocytes serve as the key progenitors of intestinal and dermal MΦs. Furthermore, a specific role for TLR13 with respect to MΦ function is supported by the response to staphylococci, where TLR13 and UNC-93B limit the cytokine response in bone marrow-derived MΦs and microglia, but not in dermal MΦs. In summary, TLR13 is a critical and site-specific receptor in the single MΦ response to ß-hemolytic streptococci.

Diffuse Unilateral Subacute Neuroretinitis Caused by Ancylostoma Hookworm.

Diffuse unilateral subacute neuroretinitis is an ocular infectious disease caused by several distinct nematodes. Definite identification of the involved nematodes is rarely achieved. We report on the molecular-based genetic identification of an Ancylostoma ceylanicum hookworm implicated in a case of diffuse unilateral subacute neuroretinitis in a child.

Omenn syndrome associated with a functional reversion due to a somatic second-site mutation in CARD11 deficiency.

Omenn syndrome (OS) is a severe immunodeficiency associated with erythroderma, lymphoproliferation, elevated IgE, and hyperactive oligoclonal T cells. A restricted T-cell repertoire caused by defective thymic T-cell development and selection, lymphopenia with homeostatic proliferation, and lack of regulatory T cells are considered key factors in OS pathogenesis. We report 2 siblings presenting with cytomegalovirus (CMV) and Pneumocystis jirovecii infections and recurrent sepsis; one developed all clinical features of OS. Both carried homozygous germline mutations in CARD11 (p.Cys150*), impairing NF-κB signaling and IL-2 production. A somatic second-site mutation reverting the stop codon to a missense mutation (p.Cys150Leu) was detected in tissue-infiltrating T cells of the OS patient. Expression of p.Cys150Leu in CARD11-deficient T cells largely reconstituted NF-κB signaling. The reversion likely occurred in a prethymic T-cell precursor, leading to a chimeric T-cell repertoire. We speculate that in our patient the functional advantage of the revertant T cells in the context of persistent CMV infection, combined with lack of regulatory T cells, may have been sufficient to favor OS. This first observation of OS in a patient with a T-cell activation defect suggests that severely defective T-cell development or homeostatic proliferation in a lymphopenic environment are not required for this severe immunopathology.

Human TLR8 senses UR/URR motifs in bacterial and mitochondrial RNA.

Toll-like receptor (TLR) 13 and TLR2 are the major sensors of Gram-positive bacteria in mice. TLR13 recognizes Sa19, a specific 23S ribosomal (r) RNA-derived fragment and bacterial modification of Sa19 ablates binding to TLR13, and to antibiotics such as erythromycin. Similarly, RNase A-treated Staphylococcus aureus activate human peripheral blood mononuclear cells (PBMCs) only via TLR2, implying single-stranded (ss) RNA as major stimulant. Here, we identify human TLR8 as functional TLR13 equivalent that promiscuously senses ssRNA. Accordingly, Sa19 and mitochondrial (mt) 16S rRNA sequence-derived oligoribonucleotides (ORNs) stimulate PBMCs in a MyD88-dependent manner. These ORNs, as well as S. aureus-, Escherichia coli-, and mt-RNA, also activate differentiated human monocytoid THP-1 cells, provided they express TLR8. Moreover, Unc93b1(-/-)- and Tlr8(-/-)-THP-1 cells are refractory, while endogenous and ectopically expressed TLR8 confers responsiveness in a UR/URR RNA ligand consensus motif-dependent manner. If TLR8 function is inhibited by suppression of lysosomal function, antibiotic treatment efficiently blocks bacteria-driven inflammatory responses in infected human whole blood cultures. Sepsis therapy might thus benefit from interfering with TLR8 function.

MyD88 in macrophages is critical for abscess resolution in staphylococcal skin infection.

When Staphylococcus aureus penetrates the epidermis and reaches the dermis, polymorphonuclear leukocytes (PMLs) accumulate and an abscess is formed. However, the molecular mechanisms that orchestrate initiation and termination of inflammation in skin infection are incompletely understood. In human myeloid differentiation primary response gene 88 (MyD88) deficiency, staphylococcal skin and soft tissue infections are a leading and potentially life-threatening problem. In this study, we found that MyD88-dependent sensing of S. aureus by dermal macrophages (Mϕ) contributes to both timely escalation and termination of PML-mediated inflammation in a mouse model of staphylococcal skin infection. Mϕs were key to recruit PML within hours in response to staphylococci, irrespective of bacterial viability. In contrast with bone marrow-derived Mϕs, dermal Mϕs did not require UNC-93B or TLR2 for activation. Moreover, PMLs, once recruited, were highly activated in an MyD88-independent fashion, yet failed to clear the infection if Mϕs were missing or functionally impaired. In normal mice, clearance of the infection and contraction of the PML infiltrate were accompanied by expansion of resident Mϕs in a CCR2-dependent fashion. Thus, whereas monocytes were dispensable for the early immune response to staphylococci, they contributed to Mϕ renewal after the infection was overcome. Taken together, MyD88-dependent sensing of staphylococci by resident dermal Mϕs is key for a rapid and balanced immune response, and PMLs are dependent on intact Mϕ for full function. Renewal of resident Mϕs requires both local control of bacteria and inflammatory monocytes entering the skin.