Sensory neurons promote immune homeostasis in the lung.

Cytokines employ downstream Janus kinases (JAKs) to promote chronic inflammatory diseases. JAK1-dependent type 2 cytokines drive allergic inflammation, and patients with JAK1 gain-of-function (GoF) variants develop atopic dermatitis (AD) and asthma. To explore tissue-specific functions, we inserted a human JAK1 GoF variant (JAK1GoF) into mice and observed the development of spontaneous AD-like skin disease but unexpected resistance to lung inflammation when JAK1GoF expression was restricted to the stroma. We identified a previously unrecognized role for JAK1 in vagal sensory neurons in suppressing airway inflammation. Additionally, expression of Calcb/CGRPß was dependent on JAK1 in the vagus nerve, and CGRPß suppressed group 2 innate lymphoid cell function and allergic airway inflammation. Our findings reveal evolutionarily conserved but distinct functions of JAK1 in sensory neurons across tissues. This biology raises the possibility that therapeutic JAK inhibitors may be further optimized for tissue-specific efficacy to enhance precision medicine in the future.

Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

BACKGROUND: Helios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans. METHODS: We performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities. RESULTS: Genome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function-repressing IL2 transcription activity-in a dominant negative manner. CONCLUSION: This study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

Heterozygous FOXN1 Variants Cause Low TRECs and Severe T Cell Lymphopenia, Revealing a Crucial Role of FOXN1 in Supporting Early Thymopoiesis.

FOXN1 is the master regulatory gene of thymic epithelium development. FOXN1 deficiency leads to thymic aplasia, alopecia, and nail dystrophy, accounting for the nude/severe combined immunodeficiency (nu/SCID) phenotype in humans and mice. We identified several newborns with low levels of T cell receptor excision circles (TRECs) and T cell lymphopenia at birth, who carried heterozygous loss-of-function FOXN1 variants. Longitudinal analysis showed persistent T cell lymphopenia during infancy, often associated with nail dystrophy. Adult individuals with heterozygous FOXN1 variants had in most cases normal CD4+ but lower than normal CD8+ cell counts. We hypothesized a FOXN1 gene dosage effect on the function of thymic epithelial cells (TECs) and thymopoiesis and postulated that these effects would be more prominent early in life. To test this hypothesis, we analyzed TEC subset frequency and phenotype, early thymic progenitor (ETP) cell count, and expression of FOXN1 target genes (Ccl25, Cxcl12, Dll4, Scf, Psmb11, Prss16, and Cd83) in Foxn1nu/+ (nu/+) mice and age-matched wild-type (+/+) littermate controls. Both the frequency and the absolute count of ETP were significantly reduced in nu/+ mice up to 3 weeks of age. Analysis of the TEC compartment showed reduced expression of FOXN1 target genes and delayed maturation of the medullary TEC compartment in nu/+ mice. These observations establish a FOXN1 gene dosage effect on thymic function and identify FOXN1 haploinsufficiency as an important genetic determinant of T cell lymphopenia at birth.

Inborn errors of immunity manifesting as atopic disorders.

Inborn errors of immunity are traditionally best known for enhancing susceptibility to infections. However, allergic inflammation, among other types of immune dysregulation, occurs frequently in patients with inborn errors of immunity. As such, the term primary atopic disorders (PADs) was recently coined to describe the group of heritable monogenic allergic disorders. It is becoming increasingly important for clinicians to recognize that allergic diseases such as food allergy, atopic dermatitis, and allergic asthma are expressions of misdirected immunity, and in patients who present with severe, early-onset, or coexisting allergic conditions, these can be indications of an underlying PAD. Identifying monogenic allergic disease through next-generation sequencing can dramatically improve outcomes by allowing the use of precision-based therapy targeting the patient's underlying molecular defect. It is therefore imperative that clinicians recognize PADs to be able to provide informed therapeutic options and improve patient outcomes. Here, we summarize the clinical features commonly seen with each of the currently known PADs, identify clinical warning signs that warrant assessment for PADs, and lastly, discuss the benefits of timely diagnosis and management of these conditions.

All hands on deck: A multidisciplinary approach to SARS-CoV-2-associated MIS-C.

Background: Multisystem Inflammatory Syndrome in Children (MIS-C) is a post-infectious complication of SARS-CoV-2 infection with overlapping features of Kawasaki disease and toxic shock syndrome. In May 2020, a provincial multidisciplinary working group was established in anticipation of emerging cases following the first wave of SARS-CoV-2 infections. Methodology: Our centre established a multidisciplinary working group for MIS-C cases in British Columbia. The group developed guidelines using the World Health Organization MIS-C case definition. Guidelines were updated using quality improvement methods as new reports and our local experience evolved. We included all children who were evaluated in person or had samples sent to our centre for MIS-C evaluation from May 2020 to April 2021. We prospectively collected patient demographics, clinical and laboratory characteristics, and treatment. Results: Fifty-two children were included. Eleven were diagnosed as confirmed MIS-C. Ten of the 11 MIS-C cases presented with shock. Gastrointestinal and mucocutaneous involvement were also prominent. Common laboratory features included elevated C-reactive protein, D-dimer, troponin, and brain natriuretic peptide. Four out of 11 (36%) had myocardial dysfunction and 3/11 (27%) had coronary artery abnormalities. All 11 patients had evidence of SARS-CoV-2 infection. Ten out of 11 (91%) received intravenous (IV) immunoglobulin and IV corticosteroids. Conclusion: Our provincial cohort of MIS-C patients were more likely to present with shock and cardiac dysfunction, require ICU admission, and be treated with corticosteroids compared to ruled out cases. Our working group's evolving process ensured children with features of MIS-C were rapidly identified, had standardized evaluation, and received appropriate treatment in our province.

Looking through the FOG: microbiome characterization and lipolytic bacteria isolation from a fatberg site.

Sewer systems are complex physical, chemical and microbial ecosystems where fats, oils and grease (FOG) present a major problem for sewer management. Their accumulation can lead to blockages ('Fatbergs'), sewer overflows and disruption of downstream wastewater treatment. Further advancements of biological FOG treatments need to be tailored to degrade the FOG, and operate successfully within the sewer environment. In this study we developed a pipeline for isolation of lipolytic strains directly from two FOG blockage sites in the UK, and isolated a range of highly lipolytic bacteria. We selected the five most lipolytic strains using Rhodamine B agar plates and pNP-Fatty acid substrates, with two Serratia spp., two Klebsiella spp. and an environmental Acinetobacter strain that all have the capacity to grow on FOG-based carbon sources. Their genome sequences identified the genetic capacity for fatty acid harvesting (lipases), catabolism and utilization (Fad genes). Furthermore, we performed a preliminary molecular characterization of the microbial community at these sites, showing a diverse community of environmental bacteria at each site, but which did include evidence of sequences related to our isolates. This study provides proof of concept to isolation strategies targeting Fatberg sites to yield candidate strains with bioremediation potential for FOG in the wastewater network. Our work sets the foundation for development of novel bioadditions tailored to the environment with non-pathogenic Acinetobacter identified as a candidate for this purpose.

Ruxolitinib as adjunctive therapy for secondary hemophagocytic lymphohistiocytosis: A case series.

INTRODUCTION: Hemophagocytic lymphohistiocytosis (HLH) is a cytokine storm syndrome associated with mortality rates of up to 88%. Standard therapy with high-dose glucocorticoids and etoposide used in adults is extrapolated from pediatric trials, with significant toxicity in older patients and those with poor performance status. The JAK1/2 inhibitor ruxolitinib has recently gained attention as a treatment option for HLH due to its broad cytokine-modulating abilities and safety profile. Herein we report our center's experience using ruxolitinib in the treatment of adult-onset secondary HLH. CASE SERIES: We report four patients with profound secondary HLH provoked by diverse triggers, including invasive pulmonary aspergillosis on background systemic lupus erythematosus, disseminated tuberculosis, and T-cell lymphoma treated with ruxolitinib as monotherapy or combination therapy in upfront and salvage settings. RESULTS: All four patients had rapid, sustained improvement in clinical status, inflammatory markers, and hematological cell counts followed by durable remission. Three patients developed manageable infectious complications postruxolitinib. CONCLUSIONS: This series demonstrates the effective use of JAK inhibition with ruxolitinib to control pathological immune activation in critically ill patients with secondary HLH and otherwise limited therapeutic options. JAK inhibition is also an area of urgent investigation for the treatment of cytokine storm associated with COVID-19.

Diverse clinical features and diagnostic delay in monogenic inborn errors of immunity: A call for access to genetic testing.

BACKGROUND: Inborn errors of immunity (IEIs) are a group of conditions affecting immune system development and function. Due to their clinical heterogeneity and lack of provider awareness, patients suffer from long diagnostic delays that increase morbidity and mortality. Next-generation sequencing facilitates earlier diagnosis and treatment of IEIs, but too often patients are unable to see the benefit of this technology due to gaps in providers' knowledge regarding which patients to test and barriers to accessing sequencing. METHODS: Here, we provide detailed clinical phenotyping and describe the impact of genetic sequencing on a cohort of 43 patients with monogenic IEIs seen in a tertiary care center from 2014 to 2019. Data were abstracted from a chart review, and a panel of clinical immunologists were consulted on the impact of genetic sequencing on their patients. RESULTS: We found that our patients had significant diagnostic delays, averaging 3.3 years; had diverse manifestations of immune system dysfunction; and had demonstrated highly complex medical needs, with on average 7.9 subspecialties involved in their care and 4.9 hospitalizations prior to definitive treatment. Our results also demonstrate the benefits of genetic testing, as it provided the majority of our patients with a diagnosis, and positively impacted their treatment, follow-up, and prognosis. CONCLUSION: This paper expands the paucity of literature on genetically confirmed IEIs in North America and supports the expansion of access to genetic testing for patients with clinical features suggesting IEI, such as those presented in our cohort.

Idiopathic splenomegaly in childhood and the spectrum of RAS-associated lymphoproliferative disease: a case report.

BACKGROUND: KRAS (KRAS proto-oncogene, GTPase; OMIM: 190,070) encodes one of three small guanosine triphosphatase proteins belonging to the RAS family. This group of proteins is responsible for cell proliferation, differentiation and inhibition of apoptosis. Gain-of-function variants in KRAS are commonly found in human cancers. Non-malignant somatic KRAS variants underlie a subset of RAS-associated autoimmune leukoproliferative disorders (RALD). RALD is characterized by splenomegaly, persistent monocytosis, hypergammaglobulinemia and cytopenia, but can also include autoimmune features and lymphadenopathy. In this report, we describe a non-malignant somatic variant in KRAS with prominent clinical features of massive splenomegaly, thrombocytopenia and lymphopenia. CASE PRESENTATION: A now-11-year-old girl presented in early childhood with easy bruising and bleeding, but had an otherwise unremarkable medical history. After consulting for the first time at 5 years of age, she was discovered to have massive splenomegaly. Clinical follow-up revealed thrombocytopenia, lymphopenia and increased polyclonal immunoglobulins and C-reactive protein. The patient had an unremarkable bone marrow biopsy, flow cytometry showed no indication of expanded double negative T-cells, while malignancy and storage disorders were also excluded. When the patient was 8 years old, whole exome sequencing performed on DNA derived from whole blood revealed a heterozygous gain-of-function variant in KRAS (NM_004985.5:c.37G > T; (p.G13C)). The variant was absent from DNA derived from a buccal swab and was thus determined to be somatic. CONCLUSIONS: This case of idiopathic splenomegaly in childhood due to a somatic variant in KRAS expands our understanding of the clinical spectrum of RAS-associated autoimmune leukoproliferative disorder and emphasizes the value of securing a molecular diagnosis in children with unusual early-onset presentations with a suspected monogenic origin.

Recurrent sterile abscesses in a case of X-linked neutropenia.

Cutaneous manifestations are common in monogenic immune disorders, including both infectious and non-infectious etiologies. We report follow-up of a case initially published in Pediatric Dermatology in 2001 of a 13-year-old boy with a history of inflammatory skin lesions and neutropenia who developed neutrophilic dermatoses precipitated by G-CSF. Whole exome sequencing performed at 36 years of age revealed a gain-of-function mutation in the WAS gene, leading to a diagnosis of X-linked neutropenia. This case report provides closure on a decades-long diagnostic odyssey and underscores the importance of genetic sequencing in patients who present with unusual dermatologic findings.

Germline CBM-opathies: From immunodeficiency to atopy.

Caspase recruitment domain (CARD) protein-B cell CLL/lymphoma 10 (BCL10)-MALT1 paracaspase (MALT1) [CBM] complexes are critical signaling adaptors that facilitate immune and inflammatory responses downstream of both cell surface and intracellular receptors. Germline mutations that alter the function of members of this complex (termed CBM-opathies) cause a broad array of clinical phenotypes, ranging from profound combined immunodeficiency to B-cell lymphocytosis. With an increasing number of patients being described in recent years, the clinical spectrum of diseases associated with CBM-opathies is rapidly expanding and becoming unexpectedly heterogeneous. Here we review major discoveries that have shaped our understanding of CBM complex biology, and we provide an overview of the clinical presentation, diagnostic approach, and treatment options for those carrying germline mutations affecting CARD9, CARD11, CARD14, BCL10, and MALT1.

DOCK8 deficiency: Insights into pathophysiology, clinical features and management.

Dedicator of cytokinesis 8 (DOCK8) deficiency is a combined immunodeficiency that exemplifies the broad clinical features of primary immunodeficiencies (PIDs), extending beyond recurrent infections to include atopy, autoimmunity and cancer. It is caused by loss of function mutations in DOCK8, encoding a guanine nucleotide exchange factor highly expressed in lymphocytes that regulates the actin cytoskeleton. Additional roles of DOCK8 have also emerged, including regulating MyD88-dependent Toll-like receptor signaling and the activation of the transcription factor STAT3. DOCK8 deficiency impairs immune cell migration, function and survival, and it impacts both innate and adaptive immune responses. Clinically, DOCK8 deficiency is characterized by allergic inflammation as well as susceptibility towards infections, autoimmunity and malignancy. This review details the pathophysiology, clinical features and management of DOCK8 deficiency. It also surveys the recently discovered combined immunodeficiency due to DOCK2 deficiency, highlighting in the process the emerging spectrum of PIDs resulting from DOCK protein family abnormalities.

Hematopoietic stem cell transplantation outcomes for 11 patients with dedicator of cytokinesis 8 deficiency.

BACKGROUND: Dedicator of cytokinesis 8 (DOCK8) deficiency can be cured by allogeneic hematopoietic stem cell transplantation (HSCT). Reports of outcomes are still limited. OBJECTIVE: We sought to analyze the results of HSCT in patients with DOCK8 deficiency and report whether approaches resulting in mixed chimerism result in clinically relevant immune reconstitution. METHODS: We performed a retrospective chart review of 11 patients with DOCK8 deficiency and measured DOCK8 expression and cytokine production. RESULTS: Of 11 patients, 7 received HSCT from related and 4 from unrelated donors; 9 patients received busulfan-based conditioning regimens. Survival was excellent (10 [91%] of 11 patients alive), including a patient who had undergone liver transplantation. Patients showed significant improvements in the frequency and severity of infections. Although eczema resolved in all, food allergies and high IgE levels persisted in some patients. Lymphopenia, eosinophilia, low numbers of naive CD8(+) T cells and switched memory B cells, and TH1/TH2 cytokine imbalance improved in most patients. Although the 8 matched related or unrelated donor recipients had full donor chimerism, all 3 recipients of mismatched unrelated donor HSCT had high levels of donor T-cell chimerism and low B-cell and myeloid cell chimerism (0% to 46%). Almost all switched memory B cells were of donor origin. All patients, including those with mixed chimerism, mounted robust antibody responses to vaccination. CONCLUSION: Allogeneic HSCT ameliorated the infectious and atopic symptoms of patients with DOCK8 deficiency. In patients with mixed chimerism, selective advantage for donor-derived T cells and switched memory B cells promoted restoration of cellular and humoral immunity and protection against opportunistic infection.

Enumeration of sulphate-reducing bacteria for assessing potential for hydrogen sulphide production in urban drainage systems.

Urban drainage structures have increasing demands which can lead to increasing hydrogen sulphide related problems forming in places where they have not previously been prevalent. This puts pressure on the methods currently used to monitor and diagnose these problems and more sophisticated methods may be needed for identifying the origin of the problems. Molecular microbiological techniques, such as quantitative polymerase chain reaction, offer a potential alternative for identifying and quantifying bacteria likely to be causing the production of hydrogen sulphide, information that, when combined with an appropriate sampling programme, can then be used to identify the potentially most effective remediation technique. The application of these methods in urban drainage systems is, however, not always simple, but good results can be achieved. In this study bacteria producing hydrogen sulphide were quantified in three small combined sewer overflow storage tanks. Bacterial counts were compared between wastewater, biofilms and sediments. Similar numbers were found in the wastewater and biofilms, with the numbers in the sediments being lower. If remediation methods for hydrogen sulphide are deemed necessary in the tanks, methods that target both the wastewater and the biofilms should therefore be considered.