Deep immunophenotyping reveals biomarkers of multisystemic inflammatory syndrome in children in a Latin American cohort.

BACKGROUND: Multisystemic inflammatory syndrome in children (MIS-C) is a life-threatening disease that occurs 2-5 weeks after severe acute respiratory syndrome coronavirus 2 exposure and is characterized by severe multisystemic inflammation. Early recognition of MIS-C is key to prognosis; therefore, establishing clinical and laboratory biomarkers that predict complications is urgently needed. OBJECTIVE: We characterized the immune response and clinical features of patients with acute MIS-C and determined biomarkers of disease in a cohort of 42 Latin American patients. METHODS: Immune characterization was performed using flow cytometry from peripheral mononuclear cells and severe acute respiratory syndrome coronavirus 2-specific humoral and cellular response was performed using flow cytometry, enzyme-linked immunospot, enzyme-linked immunosorbent assay, and neutralizing antibody assays. RESULTS: MIS-C is characterized by robust T-cell activation and cytokine storm. We uncovered that while C-X-C motif chemokine ligand (CXCL) 9, IL-10, CXCL8, CXCL10, IL-6, and IL-18 are significantly elevated in patients with shock, while CCL5 was increased in milder disease. Monocyte dysregulation was specifically associated with KD-like MIS-C. Interestingly, MIS-C patients show a natural killer cell degranulation defect that is persistent after 6 months of disease presentation, suggesting it could underlie disease susceptibility. Most MIS-C had gastrointestinal involvement, and higher levels of neopterin were identified in their stools, potentially representing a biomarker of intestinal inflammation in MIS-C. Severe acute respiratory syndrome coronavirus 2-specific cellular response and neutralizing antibodies were identifiable in convalescent MIS-C patients, suggesting sustained immunity. CONCLUSION: Clinical characterization and comprehensive immunophenotyping of Chilean MIS-C cohort provide valuable insights in understanding immune dysregulation in MIS-C and identify relevant biomarkers of disease that could be used to predict severity and organ involvement.

Autoantibodies Against Proteins Previously Associated With Autoimmunity in Adult and Pediatric Patients With COVID-19 and Children With MIS-C.

The antibody profile against autoantigens previously associated with autoimmune diseases and other human proteins in patients with COVID-19 or multisystem inflammatory syndrome in children (MIS-C) remains poorly defined. Here we show that 30% of adults with COVID-19 had autoantibodies against the lung antigen KCNRG, and 34% had antibodies to the SLE-associated Smith-D3 protein. Children with COVID-19 rarely had autoantibodies; one of 59 children had GAD65 autoantibodies associated with acute onset of insulin-dependent diabetes. While autoantibodies associated with SLE/Sjögren's syndrome (Ro52, Ro60, and La) and/or autoimmune gastritis (gastric ATPase) were detected in 74% (40/54) of MIS-C patients, further analysis of these patients and of children with Kawasaki disease (KD), showed that the administration of intravenous immunoglobulin (IVIG) was largely responsible for detection of these autoantibodies in both groups of patients. Monitoring in vivo decay of the autoantibodies in MIS-C children showed that the IVIG-derived Ro52, Ro60, and La autoantibodies declined to undetectable levels by 45-60 days, but gastric ATPase autoantibodies declined more slowly requiring >100 days until undetectable. Further testing of IgG and/or IgA antibodies against a subset of potential targets identified by published autoantigen array studies of MIS-C failed to detect autoantibodies against most (16/18) of these proteins in patients with MIS-C who had not received IVIG. However, Troponin C2 and KLHL12 autoantibodies were detected in 2 of 20 and 1 of 20 patients with MIS-C, respectively. Overall, these results suggest that IVIG therapy may be a confounding factor in autoantibody measurements in MIS-C and that antibodies against antigens associated with autoimmune diseases or other human proteins are uncommon in MIS-C.

Characterization of the Anti-Inflammatory Capacity of IL-10-Producing Neutrophils in Response to Streptococcus pneumoniae Infection.

Neutrophils are immune cells classically defined as pro-inflammatory effector cells. However, current accumulated evidence indicates that neutrophils have more versatile immune-modulating properties. During acute lung infection with Streptococcus pneumoniae in mice, interleukin-10 (IL-10) production is required to temper an excessive lung injury and to improve survival, yet the cellular source of IL-10 and the immunomodulatory role of neutrophils during S. pneumoniae infection remain unknown. Here we show that neutrophils are the main myeloid cells that produce IL-10 in the lungs during the first 48 h of infection. Importantly, in vitro assays with bone-marrow derived neutrophils confirmed that IL-10 can be induced by these cells by the direct recognition of pneumococcal antigens. In vivo, we identified the recruitment of two neutrophil subpopulations in the lungs following infection, which exhibited clear morphological differences and a distinctive profile of IL-10 production at 48 h post-infection. Furthermore, adoptive transfer of neutrophils from WT mice into IL-10 knockout mice (Il10-/- ) fully restored IL-10 production in the lungs and reduced lung histopathology. These results suggest that IL-10 production by neutrophils induced by S. pneumoniae limits lung injury and is important to mediate an effective immune response required for host survival.

Absent B cells, agammaglobulinemia, and hypertrophic cardiomyopathy in folliculin-interacting protein 1 deficiency.

Agammaglobulinemia is the most profound primary antibody deficiency that can occur due to an early termination of B-cell development. We here investigated 3 novel patients, including the first known adult, from unrelated families with agammaglobulinemia, recurrent infections, and hypertrophic cardiomyopathy (HCM). Two of them also presented with intermittent or severe chronic neutropenia. We identified homozygous or compound-heterozygous variants in the gene for folliculin interacting protein 1 (FNIP1), leading to loss of the FNIP1 protein. B-cell metabolism, including mitochondrial numbers and activity and phosphatidylinositol 3-kinase/AKT pathway, was impaired. These defects recapitulated the Fnip1-/- animal model. Moreover, we identified either uniparental disomy or copy-number variants (CNVs) in 2 patients, expanding the variant spectrum of this novel inborn error of immunity. The results indicate that FNIP1 deficiency can be caused by complex genetic mechanisms and support the clinical utility of exome sequencing and CNV analysis in patients with broad phenotypes, including agammaglobulinemia and HCM. FNIP1 deficiency is a novel inborn error of immunity characterized by early and severe B-cell development defect, agammaglobulinemia, variable neutropenia, and HCM. Our findings elucidate a functional and relevant role of FNIP1 in B-cell development and metabolism and potentially neutrophil activity.

Staphylococcus aureus small colony variants impair host immunity by activating host cell glycolysis and inducing necroptosis.

Staphylococcus aureus small colony variants (SCVs) are frequently associated with chronic infection, yet they lack expression of many virulence determinants associated with the pathogenicity of wild-type strains. We found that both wild-type S. aureus and a ΔhemB SCV prototype potently activate glycolysis in host cells. Glycolysis and the generation of mitochondrial reactive oxygen species were sufficient to induce necroptosis, a caspase-independent mechanism of host cell death that failed to eradicate S. aureus and instead promoted ΔhemB SCV pathogenicity. To support ongoing glycolytic activity, the ΔhemB SCV induced over a 100-fold increase in the expression of fumC, which encodes an enzyme that catalyses the degradatin of fumarate, an inhibitor of glycolysis. Consistent with fumC-dependent depletion of local fumarate, the ΔhemB SCV failed to elicit trained immunity and protection from a secondary infectious challenge in the skin. The reliance of the S. aureus SCV population on glycolysis accounts for much of its role in the pathogenesis of S. aureus skin infection.

Pathogenicity island excision during an infection by Salmonella enterica serovar Enteritidis is required for crossing the intestinal epithelial barrier in mice to cause systemic infection.

Pathogenicity island excision is a phenomenon that occurs in several Salmonella enterica serovars and other members of the family Enterobacteriaceae. ROD21 is an excisable pathogenicity island found in the chromosome of S. Enteritidis, S. Dublin and S. Typhi among others, which contain several genes encoding virulence-associated proteins. Excision of ROD21 may play a role in the ability of S. Enteritidis to cause a systemic infection in mice. Our previous studies have shown that Salmonella strains unable to excise ROD21 display a reduced ability to colonize the liver and spleen. In this work, we determined the kinetics of ROD21 excision in vivo in C57BL/6 mice and its effect on virulence. We quantified bacterial burden and excision frequency in different portions of the digestive tract and internal organs throughout the infection. We observed that the frequency of ROD21 excision was significantly increased in the bacterial population colonizing mesenteric lymph nodes at early stages of the infective cycle, before 48 hours post-infection. In contrast, excision frequency remained very low in the liver and spleen at these stages. Interestingly, excision increased drastically after 48 h post infection, when intestinal re-infection and mortality begun. Moreover, we observed that the inability to excise ROD21 had a negative effect on S. Enteritidis capacity to translocate from the intestine to deeper organs, which correlates with an abnormal transcription of invA in the S. Enteritidis strain unable to excise ROD21. These results suggest that excision of ROD21 is a genetic mechanism required by S. Enteritidis to produce a successful invasion of the intestinal epithelium, a step required to generate systemic infection in mice.

Cytokines in the Respiratory Airway as Biomarkers of Severity and Prognosis for Respiratory Syncytial Virus Infection: An Update.

The human respiratory syncytial virus (hRSV) is one of the most important causes of upper and lower respiratory tract infections in children and the main cause of bronchiolitis worldwide. Disease manifestations caused by hRSV may vary from mild to severe, occasionally requiring admission and hospitalization in intensive care units. Despite the high morbidity rates associated to bronchiolitis, treatment options against hRSV are limited and there are no current vaccination strategies to prevent infection. Importantly, the early identification of high-risk patients can help improve disease management and prevent complications associated with hRSV infection. Recently, the characterization of pro- and anti-inflammatory cytokine patterns produced during hRSV-related inflammatory processes has allowed the identification of potential prognosis biomarkers. A suitable biomarker should allow predicting the severity of the infection in a simple and opportune manner and should ideally be obtained from non-invasive samples. Among the cytokines associated with hRSV disease severity, IL-8, interferon-alpha (IFN-alpha), and IL-6, as well as the Th2-type cytokines thymic stromal lymphopoietin (TSLP), IL-3, and IL-33 have been highlighted as molecules with prognostic value in hRSV infections. In this review, we discuss current studies that describe molecules produced by patients during hRSV infection and their potential as biomarkers to anticipate the severity of the disease caused by this virus.

Metabolic Adaptation in Methicillin-Resistant Staphylococcus aureus Pneumonia.

Methicillin-resistant Staphylococcus aureus (MRSA) is a versatile human pathogen that is associated with diverse types of infections ranging from benign colonization to sepsis. We postulated that MRSA must undergo specific genotypic and phenotypic changes to cause chronic pulmonary disease. We investigated how MRSA adapts to the human airway to establish chronic infection, as occurs during cystic fibrosis (CF). MRSA isolates from patients with CF that were collected over a 4-year period were analyzed by whole-genome sequencing, transcriptional analysis, and metabolic studies. Persistent MRSA infection was associated with staphylococcal metabolic adaptation, but not changes in immunogenicity. Adaptation was characterized by selective use of the tricarboxylic acid cycle cycle and generation of biofilm, a means of limiting oxidant stress. Increased transcription of specific metabolic genes was conserved in all host-adapted strains, most notably a 10,000-fold increase in fumC, which catalyzes the interconversion of fumarate and malate. Elevated fumarate levels promoted in vitro biofilm production in clinical isolates. Host-adapted strains preferred to assimilate glucose polymers and pyruvate, which can be metabolized to generate N-acetylglucosamine polymers that comprise biofilm. MRSA undergoes substantial metabolic adaptation to the human airway to cause chronic pulmonary infection, and selected metabolites may be useful therapeutically to inhibit infection.

Interleukin-10 Produced by Myeloid-Derived Suppressor Cells Provides Protection to Carbapenem-Resistant Klebsiella pneumoniae Sequence Type 258 by Enhancing Its Clearance in the Airways.

Carbapenem-resistant Klebsiella pneumoniae sequence type 258 (CRKP-ST258) can cause chronic infections in lungs and airways, with repeated episodes of bacteremia. In this report we addressed whether the recruitment of myeloid cells producing the anti-inflammatory cytokine interleukin-10 (IL-10) modulates the clearance of CKRP-ST258 in the lungs and establishes bacterial persistence. Our data demonstrate that during pneumonia caused by a clinical isolate of CRKP-ST258 (KP35) there is an early recruitment of monocyte-myeloid-derived suppressor cells (M-MDSCs) and neutrophils that actively produce IL-10. However, M-MDSCs were the cells that sustained the production of IL-10 over the time of infection evaluated. Using mice unable to produce IL-10 (IL-10-/-), we observed that the production of this cytokine during the infection caused by KP35 is important to control bacterial burden, to prevent lung damage, to modulate cytokine production, and to improve host survival. Importantly, intranasal transfer of bone marrow-derived M-MDSCs from mice able to produce IL-10 at 1 day prior to infection improved the ability of IL-10-/- mice to clear KP35 in the lungs, decreasing their mortality. Altogether, our data demonstrate that IL-10 produced by M-MDSCs is required for bacterial clearance, reduction of lung tissue damage, and host survival during KP35 pneumonia.

Expanding the Current Knowledge About the Role of Interleukin-10 to Major Concerning Bacteria.

Interleukin-10 (IL-10) is one of the most important anti-inflammatory cytokine produced during bacterial infection. Two related phenomena explain the importance of IL-10 production in this context: first, the wide range of cells able to produce this cytokine and second, the wide effects that it causes on target cells. In a previous report we described opposing roles of IL-10 production during bacterial infection. Overall, during infections caused by intracellular bacteria or by pathogens that modulate the inflammatory response, IL-10 production facilitates bacterial persistence and dissemination within the host. Whereas during infections caused by extracellular or highly inflammatory bacteria, IL-10 production reduces host tissue damage and facilitates host survival. Given that these data were obtained using antibiotic susceptible bacteria, the potential application of these studies to multi-drug resistant (MDR) bacteria needs to be evaluated. MDR bacteria can become by 2050 a major death cause worldwide, not only for its ability to resist antimicrobial therapy but also because the virulence of these strains is different as compared to antibiotic susceptible strains. Therefore, it is important to understand the interaction of MDR-bacteria with the immune system during infection. This review discusses the current data about the role of IL-10 during infections caused by major circulating antibiotic resistant bacteria. We conclude that the production of IL-10 improves host survival during infections caused by extracellular or highly inflammatory bacteria, however, it is detrimental during infections caused by intracellular bacteria or bacterial pathogens that modulate the inflammatory response. Importantly, during MDR-bacterial infections a differential IL-10 production has been described, compared to non-MDR bacteria, which might be due to virulence factors specific of MDR bacteria that modulate production of IL-10. This knowledge is important for the development of new therapies against infections caused by these bacteria, where antibiotics effectiveness is dramatically decreasing.