Serum cytokine panels in pediatric clinical practice.

BACKGROUND: Cytokines are soluble signaling proteins that regulate inflammation and coordinate immune responses. Serum cytokine panels are increasingly used in medical practice, yet our understanding of cytokines as biomarkers for disease remains limited. OBJECTIVE: We sought to analyze real-world single-center use of a multiplexed cytokine panel, correlate its results with diagnosis and severity, and explore its use in pediatric practice. METHODS: A multiplexed cytokine panel, able to return same-day results, was implemented in April 2020 at the Children's Hospital of Philadelphia (Philadelphia, Pa) and its performance was validated for clinical use. Coded patient data were collected using the REDCap database, and correlations between cytokine levels and outcomes of interest were analyzed retrospectively. RESULTS: Cytokine levels correlate with acuity of care, with patients admitted to the pediatric intensive care unit having the highest cytokine values. Patients with familial hemophagocytic lymphohistiocytosis (fHLH) showed prominent peaks in IFN-γ, IL-10, and TNF, whereas patients with sepsis exhibited high IL-6 and IL-8 with relatively modest IFN-γ. Cytokine release syndrome (CRS) after chimeric antigen receptor T-cell therapy often demonstrated pan-panel positivity at peak levels, with a similar pattern as that of fHLH. A ratio of [IFN-γ] + [IL-10]/[IL-6] + [IL-8] levels was able to distinguish fHLH and CRS from severe sepsis. CONCLUSIONS: Cytokine levels correlate with severity of illness and can help differentiate between syndromes that present similarly, including fHLH and CRS compared with sepsis. Cytokine panels can be used as biomarkers to inform diagnosis and management decisions, but significant work remains to dissect complex clinical patterns of disease.

Hyperferritinemia Screening to Aid Identification and Differentiation of Patients with Hyperinflammatory Disorders.

High ferritin is an important and sensitive biomarker for the various forms of hemophagocytic lymphohistiocytosis (HLH), a diverse and deadly group of cytokine storm syndromes. Early action to prevent immunopathology in HLH often includes empiric immunomodulation, which can complicate etiologic work-up and prevent collection of early/pre-treatment research samples. To address this, we instituted an alert system at UPMC Children's Hospital where serum ferritin > 1000 ng/mL triggered real-time chart review, assessment of whether the value reflected "inflammatory hyperferritnemia (IHF)", and biobanking of remnant samples from consenting IHF patients. We extracted relevant clinical data; periodically measured serum total IL-18, IL-18 binding protein (IL-18BP), and CXCL9; retrospectively classified patients by etiology into infectious, rheumatic, or immune dysregulation; and subjected a subgroup of samples to a 96-analyte biomarker screen. 180 patients were identified, 30.5% of which had IHF. Maximum ferritin levels were significantly higher in patients with IHF than with either hemoglobinopathy or transplant, and highly elevated total IL-18 levels were distinctive to patients with Stills Disease and/or Macrophage Activation Syndrome (MAS). Multi-analyte analysis showed elevation in proteins associated with cytotoxic lymphocytes in all IHF samples when compared to healthy controls and depression of proteins such as ANGPT1 and VEGFR2 in samples from hyperferritinemic sepsis patients relative to non-sepsis controls. This real-time IFH screen proved feasible and efficient, validated prior observations about the specificity of IL-18, enabled early sample collection from a complex population, suggested a unique vascular biomarker signature in hyperferritinemic sepsis, and expanded our understanding of IHF heterogeneity.

The RAG1 Ubiquitin Ligase Domain Stimulates Recombination of TCRß and TCRα Genes and Influences Development of αß T Cell Lineages.

RAG1/RAG2 (RAG) endonuclease-mediated assembly of diverse lymphocyte Ag receptor genes by V(D)J recombination is critical for the development and immune function of T and B cells. The RAG1 protein contains a ubiquitin ligase domain that stabilizes RAG1 and stimulates RAG endonuclease activity in vitro. We report in this study that mice with a mutation that inactivates the Rag1 ubiquitin ligase in vitro exhibit decreased rearrangements and altered repertoires of TCRß and TCRα genes in thymocytes and impaired thymocyte developmental transitions that require the assembly and selection of functional TCRß and/or TCRα genes. These Rag1 mutant mice present diminished positive selection and superantigen-mediated negative selection of conventional αß T cells, decreased genesis of invariant NK T lineage αß T cells, and mature CD4+ αß T cells with elevated autoimmune potential. Our findings reveal that the Rag1 ubiquitin ligase domain functions in vivo to stimulate TCRß and TCRα gene recombination and influence differentiation of αß T lineage cells, thereby establishing replete diversity of αß TCRs and populations of αß T cells while restraining generation of potentially autoreactive conventional αß T cells.

Cytokines in Cytokine Storm Syndrome.

As the eponymous mediators of the cytokine storm syndrome, cytokines are a pleomorphic and diverse set of soluble molecules that activate or suppress immune functions in a wide variety of ways. The relevant cytokines for each CSS are likely a result of differing combinations of environmental triggers and host susceptibilities. Because cytokines or their receptors may be specifically targeted by biologic therapeutics, understanding which cytokines are relevant for disease initiation and propagation for each unique CSS is of major clinical importance. This chapter will review what is known about the role of cytokines across the spectrum of CSS.

Anti-Interferon-γ Therapy for Cytokine Storm Syndromes.

A vast body of evidence provides support to a central role of exaggerated production of interferon-γ (IFN-γ) in causing hypercytokinemia and signs and symptoms of hemophagocytic lymphohistiocytosis (HLH). In this chapter, we will describe briefly the roles of IFN-γ in innate and adaptive immunity and in host defense, summarize results from animal models of primary HLH and secondary HLH with particular emphasis on targeted therapeutic approaches, review data on biomarkers associated with activation of the IFN-γ pathway, and discuss initial efficacy and safety results of IFN-γ neutralization in humans.

Introduction.

The coronavirus disease 2019 (COVID-19) pandemic emerged just months after the publication of the first ever textbook devoted to cytokine storm syndromes (CSSs). The severe disease caused by COVID-19 and the intersection between immune responses and their pathologies played out before the world in media reports, in scientific publications, and through the personal narratives of millions of people's experiences. An entirely new immune-mediated disease, multisystem inflammatory disease in children (MISC), was described. Cytokines played a role in all of these areas, bringing the idea of a cytokine storm squarely to the front and center of the public eye. At the same time, science continued to progress in the lab and in the clinic, thus illuminating our understanding of CSSs both old and new since the publication of the first edition of this book. It was clear that a new edition was needed to keep up with these changes.

Improvement of Refractory Systemic Juvenile Idiopathic Arthritis-Associated Lung Disease with Single-Agent Blockade of IL-1ß and IL-18.

Systemic juvenile idiopathic arthritis associated with interstitial lung disease (SJIA-LD) represents a highly morbid subset of SJIA for which effective therapies are lacking. We report the case of a patient with refractory SJIA-LD who underwent treatment with MAS-825, an investigational bispecific monoclonal antibody targeting IL-1ß and IL-18. MAS-825 treatment was associated with a marked reduction in total IL-18 and free IL-18 in both serum and bronchoalveolar lavage fluid (BAL). Baseline oxygen saturation, exercise tolerance, and quality of life metrics improved after treatment with MAS-825, while pulmonary function testing remained stable. Following treatment, the BAL showed no evidence of pulmonary alveolar proteinosis and inflammatory infiltrates were markedly reduced, reflected by decreased numbers of CD4 T-cells, CD8 T-cells, and macrophages. The patient was able to wean entirely off systemic corticosteroids and other biologics after 10 months of treatment with MAS-825 and experienced no side effects of the drug. This case demonstrates improvement in pulmonary symptoms, lung inflammation, and burden of immunomodulatory therapy after treatment with MAS-825 and suggests that simultaneous targeting of both IL-1ß and IL-18 may be a safe and effective treatment strategy in SJIA-LD.

The 2022 EULAR/ACR points to consider at the early stages of diagnosis and management of suspected haemophagocytic lymphohistiocytosis/macrophage activation syndrome (HLH/MAS).

OBJECTIVE: Haemophagocytic lymphohistiocytosis (HLH) and macrophage activation syndrome (MAS) are life-threatening systemic hyperinflammatory syndromes that can develop in most inflammatory contexts. They can progress rapidly, and early identification and management are critical for preventing organ failure and mortality. This effort aimed to develop evidence-based and consensus-based points to consider to assist clinicians in optimising decision-making in the early stages of diagnosis, treatment and monitoring of HLH/MAS. METHODS: A multinational, multidisciplinary task force of physician experts, including adult and paediatric rheumatologists, haematologist/oncologists, immunologists, infectious disease specialists, intensivists, allied healthcare professionals and patients/parents, formulated relevant research questions and conducted a systematic literature review (SLR). Delphi methodology, informed by SLR results and questionnaires of experts, was used to generate statements aimed at assisting early decision-making and optimising the initial care of patients with HLH/MAS. RESULTS: The task force developed 6 overarching statements and 24 specific points to consider relevant to early recognition of HLH/MAS, diagnostic approaches, initial management and monitoring of HLH/MAS. Major themes included the simultaneous need for prompt syndrome recognition, systematic evaluation of underlying contributors, early intervention targeting both hyperinflammation and likely contributors, careful monitoring for progression/complications and expert multidisciplinary assistance. CONCLUSION: These 2022 EULAR/American College of Rheumatology points to consider provide up-to-date guidance, based on the best available published data and expert opinion. They are meant to help guide the initial evaluation, management and monitoring of patients with HLH/MAS in order to halt disease progression and prevent life-threatening immunopathology.

The Liver in Hemophagocytic Lymphohistiocytosis: Not an Innocent Bystander.

Hemophagocytic lymphohistiocytosis (HLH) is a rare multisystemic hyperinflammatory disease commonly associated with hepatic dysfunction. Liver injury is mediated by unchecked antigen presentation, hypercytokinemia, dysregulated cytotoxicity by natural killer and CD8 T cells, and disruption of intrinsic hepatic metabolic pathways. Over the past decade, there have been significant advances in diagnostics and expansion in therapeutic armamentarium for this disorder allowing for improved morbidity and mortality. This review discusses the clinical manifestations and pathogenesis of HLH hepatitis in both familial and secondary forms. It will review growing evidence that the intrinsic hepatic response to hypercytokinemia in HLH perpetuates disease progression and the novel therapeutic approaches for patients with HLH-hepatitis/liver failure.

Adenovirus is Not Detected in Liver Tissue From a Historical Multicenter Cohort of Children With Acute Liver Failure.

There has been a recent surge in cases of pediatric acute hepatitis and pediatric acute liver failure (PALF) of unknown cause. Several reports have described clusters of these children who were positive for adenovirus (AdV) DNA, primarily in peripheral blood but some in liver tissue. We tested archived liver tissue specimens from a historical cohort of 44 children with PALF who were enrolled in a multicenter biorepository between 2007 and 2014 for AdV 40/41 using quantitative polymerase chain reaction. Most children had final diagnosis indeterminate. All samples were negative. Our findings suggest that AdV was unlikely to be an unidentified cause of indeterminate PALF during this past era. The significance of AdV viremia in contemporary cohorts of children with PALF remains unknown and requires further study.

Lymphocytes Utilize Somatic Mutations, Epigenetic Silencing, and the Proteasome to Escape Truncated WASP Expression.

Wiskott-Aldrich Syndrome Protein (WASP) deficiency causes Wiskott-Aldrich Syndrome (WAS), a sex-linked disorder characterized by combined immunodeficiency, microthrombocytopenia, and eczema. Like WASP-deficient humans, WASP-deficient mice produce normal numbers of functionally defective T cells. Here, we report a WAS patient with a novel germline frameshifting WAS mutation encoding a truncated form of WASP lacking the C-terminal cofilin homology (C) and the acidic region (A) domains (WASPΔCA). Although stably overexpressed in embryonic kidney cell lines, WASPΔCA was undetectable in circulating patient leukocytes. Deep sequencing, transcript profiling, and protein degradation analyses demonstrated patient lymphocytes employ an array of genetic, epigenetic, and proteasomal strategies to avoid expressing WASPΔCA.

Ruxolitinib: Targeted Approach for Treatment of Autoinflammatory Very Early Onset Inflammatory Bowel Disease.

Very early onset inflammatory bowel disease (VEO-IBD), diagnosed <6 years old, can be genetically and phenotypically distinct and more refractory than older-onset IBD. Identified causal monogenic defects have been targeted therapeutically in a small subset of VEO-IBD1; however, for most of these children, treatment strategies, such as phenotypic profiles, are critically needed to improve outcomes.

Hematologic abnormalities in Aicardi Goutières Syndrome.

BACKGROUND: Because of the broad clinical spectrum, heritable autoinflammatory diseases present a management and therapeutic challenge. The most common genetic interferonopathy, Aicardi Goutières Syndrome (AGS), is associated with early onset neurologic disability and systemic inflammation. The chronic inflammation of AGS is the result of dysregulation of interferon (IFN) expression by one of nine genes within converging pathways. While each AGS subtype shares common features, distinct patterns of severity and potential for systemic complications amongst the genotypes are emerging. Multilineage cytopenias are a potentially serious, but poorly understood, complication of AGS. As immunomodulatory treatment options are developed, it is important to characterize the role of the disease versus treatment in hematologic abnormalities. This will allow for better understanding and management of cytopenia. METHODS: In total, 142 individuals with molecularly-confirmed AGS were included. Information on genotype, demographics, and all available hematologic laboratory values were collected from existing medical records. As part of a clinical trial, a subset of this cohort (n = 52) were treated with a janus kinase inhibitor (baricitinib), and both pre- and post-treatment values were included. Abnormal values were graded based on Common Terminology Criteria for Adverse Events (CTCAE v5.0), supplemented with grading definitions for thrombocytosis, and were compared across genotypes and baricitinib exposure. RESULTS: In total, 11,184 laboratory values were collected over a median of 2.54 years per subject (range 0-22.68 years). To reduce bias from repeated sampling within a limited timeframe, laboratory results were restricted to the most abnormal value within a month (n = 8485). The most common abnormalities were anemia (noted in 24% of subjects prior to baricitinib exposure), thrombocytopenia (9%), and neutropenia (30%). Neutropenia was most common in the SAMHD1 cohort and increased with baricitinib exposure (38/69 measurements on baricitinib versus 14/121 while not on baricitinib). Having an abnormality prior to treatment was associated with having an abnormality on treatment for neutropenia and thrombocytopenia. CONCLUSION: By collecting available laboratory data throughout the lifespan, we were able to identify novel patterns of hematologic abnormalities in AGS. We found that AGS results in multilineage cytopenias not limited to the neonatal period. Neutropenia, anemia, and thrombocytopenia were common. Moderate-severe graded events of neutropenia, anemia, and leukopenia were more common on baricitinib, but rarely of clinical consequence. Based on these results, we would recommend careful monitoring of hematologic parameters of children affected by AGS throughout the lifespan, especially while on therapy, and consideration of AGS as a potential differential diagnosis in children with neurologic impairment of unclear etiology with hematologic abnormalities. Trial registration ClinicalTrials.gov Identifier: NCT01724580 ClinicalTrials.gov Identifier: NCT03921554.

IFNs Drive Development of Novel IL-15-Responsive Macrophages.

Disruption in homeostasis of IL-15 is linked to poor maternal and fetal outcomes during pregnancy. The only cells described to respond to IL-15 at the early maternal-fetal interface have been NK cells. We now show a novel population of macrophages, evident in several organs but enriched in the uterus of mice and humans, expressing the ß-chain of the IL-15R complex (CD122) and responding to IL-15. CD122+ macrophages (CD122+Macs) are morphologic, phenotypic, and transcriptomic macrophages that can derive from bone marrow monocytes. CD122+Macs develop in the uterus and placenta with kinetics that mirror IFN activity at the maternal-fetal interface. M-CSF permits macrophages to express CD122, and IFNs are sufficient to drive expression of CD122 on macrophages. Neither type I nor type II IFNs are required to generate CD122+Macs, however. In response to IL-15, CD122+Macs activate the ERK signaling cascade and enhance production of proinflammatory cytokines after stimulation with the TLR9 agonist CpG. Finally, we provide evidence of human cells that phenocopy murine CD122+Macs in secretory phase endometrium during the implantation window and in first-trimester uterine decidua. Our data support a model wherein IFNs local to the maternal-fetal interface direct novel IL-15-responsive macrophages with the potential to mediate IL-15 signals critical for optimal outcomes of pregnancy.

Human Adenovirus 7-Associated Hemophagocytic Lymphohistiocytosis-like Illness: Clinical and Virological Characteristics in a Cluster of Five Pediatric Cases.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening condition of immune dysregulation. Children often suffer from primary genetic forms of HLH, which can be triggered by infection. Others suffer from secondary HLH as a complication of infection, malignancy, or rheumatologic disease. Identifying the exact cause of HLH is crucial, as definitive treatment for primary disease is hematopoietic stem cell transplant. Adenoviruses have been associated with HLH but molecular epidemiology data are lacking. METHODS: We describe the clinical and virologic characteristics of 5 children admitted with adenovirus infection during 2018-2019 who developed HLH or HLH-like illness. Detailed virologic studies, including virus isolation and comprehensive molecular typing were performed. RESULTS: All patients recovered; clinical management varied but included immunomodulating and antiviral therapies. A genetic predisposition for HLH was not identified in any patient. Adenovirus isolates were recovered from 4/5 cases; all were identified as genomic variant 7d. Adenovirus type 7 DNA was detected in the fifth case. Phylogenetic analysis of genome sequences identified 2 clusters-1 related to strains implicated in 2016-2017 outbreaks in Pennsylvania and New Jersey, the other related to a 2009 Chinese strain. CONCLUSIONS: It can be challenging to determine whether HLH is the result of an infectious pathogen alone or genetic predisposition triggered by an infection. We describe 5 children from the same center presenting with an HLH-like illness after onset of adenovirus type 7 infection. None of the patients were found to have a genetic predisposition to HLH. These findings suggest that adenovirus 7 infection alone can result in HLH.

Diagnostic Challenges in Pediatric Hemophagocytic Lymphohistiocytosis.

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome of severe immune dysregulation that encompasses a broad range of underlying genetic diseases and infectious triggers. Monogenic conditions, autoimmune diseases, and infections can all drive the phenotype of HLH and associated immune hyperactivation with hypercytokinemia. A diagnosis of HLH usually requires a combination of clinical and laboratory findings; there is no single sensitive and specific diagnostic test, which often leads to "diagnostic dilemmas" and delays in treatment initiation. Ferritin levels, one of the most commonly used screening tests, were collected across a large tertiary care pediatric hospital to identify the positive predictive value for HLH. Herein, we present several cases that illustrate the clinical challenges of confirming an HLH diagnosis. Additionally, we report on the utility of establishing a formal multi-disciplinary group to aid the prompt diagnosis and treatment of patients presenting with HLH-like pathophysiologies.

Muscularis macrophage development in the absence of an enteric nervous system.

The nervous system of the bowel regulates the inflammatory phenotype of tissue resident muscularis macrophages (MM), and in adult mice, enteric neurons are the main local source of colony stimulating factor 1 (CSF1), a protein required for MM survival. Surprisingly, we find that during development MM colonize the bowel before enteric neurons. This calls into question the requirement for neuron-derived CSF1 for MM colonization of the bowel. To determine if intestinal innervation is required for MM development, we analyzed MM of neonatal Ret-/- (Ret KO) mice that have no enteric nervous system in small bowel or colon. We found normal numbers of well-patterned MM in Ret KO bowel. Similarly, the abundance and distribution of MM in aganglionic human colon obtained from Hirschsprung disease patients was normal. We also identify endothelial cells and interstitial cells of Cajal as the main sources of CSF1 in the developing bowel. Additionally, MM from neonatal Ret KOs do not differ from controls in baseline activation status or cytokine-production in response to lipopolysaccharide. Unexpectedly, these data demonstrate that the enteric nervous system is dispensable for MM colonization and patterning in the bowel, and suggest that modulatory interactions between MM and the bowel nervous system are established postnatally.

Sterile particle-induced inflammation is mediated by macrophages releasing IL-33 through a Bruton's tyrosine kinase-dependent pathway.

Initiation of the innate sterile inflammatory response that can develop in response to microparticle exposure is little understood. Here, we report that a potent type 2 immune response associated with the accumulation of neutrophils, eosinophils and alternatively activated (M2) macrophages was observed in response to sterile microparticles similar in size to wear debris associated with prosthetic implants. Although elevations in interleukin-33 (IL-33) and type 2 cytokines occurred independently of caspase-1 inflammasome signalling, the response was dependent on Bruton's tyrosine kinase (BTK). IL-33 was produced by macrophages and BTK-dependent expression of IL-33 by macrophages was sufficient to initiate the type 2 response. Analysis of inflammation in patient periprosthetic tissue also revealed type 2 responses under aseptic conditions in patients undergoing revision surgery. These findings indicate that microparticle-induced sterile inflammation is initiated by macrophages activated to produce IL-33. They further suggest that both BTK and IL-33 may provide therapeutic targets for wear debris-induced periprosthetic inflammation.

Convalescent plasma for pediatric patients with SARS-CoV-2-associated acute respiratory distress syndrome.

There are no proven safe and effective therapies for children who develop life-threatening complications of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Convalescent plasma (CP) has demonstrated potential benefit in adults with SARS-CoV-2, but has theoretical risks.We present the first report of CP in children with life-threatening coronavirus disease 2019 (COVID-19), providing data on four pediatric patients with acute respiratory distress syndrome. We measured donor antibody levels and recipient antibody response prior to and following CP infusion. Infusion of CP was not associated with antibody-dependent enhancement (ADE) and did not suppress endogenous antibody response. We found CP was safe and possibly efficacious. Randomized pediatric trials are needed.

Activated CD8 T-cell Hepatitis in Children With Indeterminate Acute Liver Failure: Results From a Multicenter Cohort.

OBJECTIVES: In many pediatric acute liver failure (PALF) cases, a diagnosis is not identified, and the etiology is indeterminate (IND-PALF). Our pilot study found dense CD8 T-cell infiltrates and increased T-cell clonality in liver specimens from IND-PALF patients. We aimed to validate these findings in a multicenter cohort with investigators blinded to diagnosis. METHODS: PALF Study Group registry subjects with IND-PALF (n = 37) and known diagnoses (DX-PALF) (n = 18), ages 1 to 17 years, with archived liver tissue were included. Liver tissue slides were stained for T cells (CD8 and CD4), B cells (CD20), macrophages (CD163), perforin, and tissue resident-memory T cells (Trm, CD103), and scored as minimal, moderate, or dense. Lymphocytes were isolated from frozen liver tissue for T-cell receptor beta (TCRß) sequencing. RESULTS: Dense hepatic CD8 staining was found in significantly more IND-PALF (n = 29, 78%) compared with DX-PALF subjects (n = 5, 28%) (P = 0.001). IND-PALF subjects were more likely to have dense or moderate perforin (88% vs 50%, P = 0.03) and CD103 (82% vs 40%, P = 0.02) staining compared with DX-PALF subjects. TCRß sequencing of 15 IND-PALF cases demonstrated increased clonal overlap compared with 6 DX-PALF cases (P = 0.002). CONCLUSIONS: Dense infiltration of effector Trm CD8 T cells characterizes liver tissue from IND-PALF subjects. Increased clonality suggests the T-cell expansion is antigen(s)-driven as opposed to a nonspecific inflammatory response. These findings support CD8 staining as a new biomarker of the activated CD8 T-cell PALF phenotype. Future studies are needed to characterize potential antigens, host risk factors, and inflammatory pathways with the goal of developing targeted therapies.