key: cord-0699163-belyo5a6
authors: García-Salido, Alberto; Cuenca-Carcelén, Sandra; Castillo-Robleda, Ana
title: CD64, CD11a and CD18 leukocytes expression in children with SARS-CoV-2 multisystem inflammatory syndrome versus children with Kawasaki disease: Similar but not the same
date: 2021-01-22
journal: Med Clin (Engl Ed)
DOI: 10.1016/j.medcle.2020.09.002
sha: a4912123567708548cba4f77d6c033ecd2b74904
doc_id: 699163
cord_uid: belyo5a6

nan

The immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) appears to be a critical factor in the prognosis of coronavirus disease 2019 (COVID-19). 1 Generally, children are less affected and developed asymptomatic or mild forms. Despite this, pediatricians across Europe have describe severe cases of the disease. Firstly recognized as "Kawasaki like" processes and later named as pediatric multisystem syndrome temporally associated with SARS-CoV-2 (PIMS-TS). [2] [3] [4] We have seen similar cases in our center adding to its clinical and analytical study the application of immunophenotyping by flow cytometry (FC). 4 In this report, we describe CD64, CD18, and CD11a expression in three children with PIMS-TS and compare it with three cases of Kawasaki Disease (KD, years 2018 and 2019). The CD64 is a type I high-affinity receptor for the Fc fraction of the immunoglobulin G, located on the surface of monocytes, macrophages, dendritic cells, and neutrophils. Increased CD64 on the cell surface is related to the intensity of stimulation received by inflammatory cytokines. Additionally, CD18, also known as integrin ␤2, participates in leukocyte adhesion and signaling. CD11a associates with CD18 to form the lymphocyte function-associated antigen 1, or LFA-1. Expressed on leukocytes, this T cell integrin plays a central role in leukocyte cell-cell interactions and lymphocyte stimulation.

The cases clinical trajectories are described in Table 1 . The children were studied after informed consent obtained from their parents or legal guardians. The samples were collected in sterile EDTA at room temperature, refrigerated at 4 • C and analyzed by FC within 24 h. Cell surface expression of CD64, CD18, and CD11a were measured by BD FACS Canto II flow cytometer (Becton Dickinson, New York, USA). CD64 (clone 10.1), CD18 (clone CBR LFA-1/2), and CD11a (clone HI111) monoclonal antibodies were obtained from Biolegend ® (San Diego, CA, USA). Expressions were measured in monocytes, neutrophils and lymphocytes were identified on a dot-plot and gated. Cell viability was confirmed by 7-AAD staining. At least 10,000 events were recorded for each sample. Flow-cytometry settings and samples were prepared according to manufacturer instructions. The intensity of CD64, CD18, and CD11a surface expression were measured as mean fluorescence intensity in arbitrary units (MFI).

The expression of CD64, CD11a, and CD18 are in Table 1 . All PIMS-TS cases received methylprednisolone prior to FC, the KD cases were studied before received immunoglobulin. As main finding, we observe higher upregulation of the three proteins studied in SARS-CoV-2 patients. This response appears to be similar but higher than in KD.

Recent papers have described that a dysregulated immune response may result in inflammation and clinical worsening in COVID-19 cases. Our cases show high levels of CD64 expression. This expression is also higher than the described in some KD Laboratory criteria: 1 = CRP > 3 mg/dl, 2 = ESR > 40 mm/h, 3 = WBC count >15,000/mm 3 ; 4 = normocytic anemia for age; 5 = platelets after fever for 7 d > 450,000/mm 3 ; 6 = ALT >45 IU/L; 7 = albumin < 3.0 g/dL; and 8 = urine >10 WBCs/high-power field. Legend and experiment data: The children were studied after informed consent obtained from their parents or legal guardians. The samples obtained were collected in sterile EDTA at room temperature or refrigerated at 4 • C and analyzed by FC within 24 h. Cell surface expression of CD64, CD18, and CD11a was measured by BD FACS Canto II flow cytometer (Becton Dickinson, New York, USA). CD64 (clone 10.1), CD18 (clone CBR LFA-1/2), and CD11a (clone HI111) monoclonal antibodies were obtained from Biolegend ® (San Diego, CA, USA). Expressions were measured in monocytes, neutrophils, and lymphocytes. Cell viability was confirmed by 7-AAD staining. At least 10,000 events were recorded for each sample. Flow-cytometry settings and samples were prepared according to manufacturer instructions. Neutrophils, monocytes, and lymphocytes were identified on a dot-plot and gated. The intensity of CD64, CD18, and CD11a surface expression were measured as mean fluorescence intensity in arbitrary units (MFI).

autoinflammatory diseases, so it could indicate immune dysregulation. Cytokines such as IL-1b, IL-6, and TNF-a affect the presence of CD64 on the cell surface. 5 Regarding the LFA-1, it is known that plays a key role in leukocyte migration into tissues. Lymphopenia is a common finding in COVID-19 patients. 1 This situation may be linked to the migration of CD8+ lymphocytes to the infected tissues. As seen in Table 1 , the CD11a upregulation in CD8+ is clear and could be linked to this process. Additionally, LFA-1 is involved in the process of cytotoxic T cell-mediated killing as well as antibody-mediated killing by granulocytes and monocytes. We observed an exacerbated CD8+ LFA-1 expression compared to KD cases.

The nonantimicrobial COVID-19 therapies proposed are intended to downregulate the immune system. The cytokine storm theory, coupled with analytical data that suggest immune dysregulation or macrophage activation syndrome justify these therapies. In our cases, all FC analyses were performed after almost four days following disease onset. Also, in one case FC was conducted in a patient with positive immunoglobulin G to SARS-CoV-2, which is not a precocious immune reaction. 1 The observation of high CD64 expression helped to detect a proinflammatory status and helped to take the decision of using immunoregulatory therapies. 4 In summary, we compare for the first time the immunophenotype of children with severe SARS-CoV-2 infection versus children with KD. We observed significant but higher upregulation of CD64, CD18, and CD11a expression on leukocytes. Prospective studies with a higher number of cases should be conducted to confirm this observation.

This study was partially funded by "Fundación Familia Alonso".

No conflict interest.

Dysregulation of immune response in patients with coronavirus 2019 (COVID-19) in Wuhan, China

An outbreak of severe Kawasaki-like disease at the Italian epicentre of the SARS-CoV-2 epidemic: an observational cohort study

Hyperinflammatory shock in children during COVID-19 pandemic

Severe SARS-CoV-2 infection in children with suspected acute abdomen: a case series from a tertiary hospital in Spain

Markedly elevated CD64 expressions on neutrophils and monocytes are useful for diagnosis of periodic fever, aphthous stomatitis, pharyngitis, and cervical adenitis (PFAPA) syndrome during flares

Tumor necrosis factor receptor associated periodic syndrome: Phenotypic heterogeneity in a family carrying a low-penetrance mutation ଝ Síndrome periódico asociado al receptor del factor de necrosis tumoral: heterogeneidad clínica en una familia con mutación de baja penetranciaTo the Editor:Hereditary periodic fever syndromes are a group of diseases caused by a dysregulation of the innate immune system, characterized by recurrent episodes of fever and systemic inflammatory signs, alternating with relatively symptom-free periods. These include the tumour necrosis factor receptor-associated periodic syndrome (TRAPS).TRAPS is an autosomal dominant disease that affects one in every million inhabitants, caused by the TNFR1A gene mutation. It is characterized by recurrent flares of fever, myalgia, migratory rash, and ocular involvement. 1, 2 The diagnosis is clinical and genetic. It often occurs during childhood, although there are cases of late onset. 1,2 Secondary amyloidosis is the most feared complication.

Affected man Asymptomatic carrier Our study reports the case of a family of 4 members who carry the R92Q mutation of the gene TNFR1A.The index case was a 41-year-old woman who, since the age of 14, had suffered from recurrent flares of fever lasting more than a week, general malaise, sterile oligoarthritis, myalgias, migratory salmon coloured skin rash, painful on palpation and bilateral eyelid oedema, with poor response to non-steroidal anti-inflammatory drugs, corticosteroids or colchicine. During these exacerbations, the patient had elevated acute phase reactants, which went back to normal values during the symptom-free periods. A brother of the patient had similar episodes, but of less intensity and onset in adulthood.With the suspected diagnosis of hereditary autoinflammatory syndrome, a genetic analysis was performed, detecting an heterozygous R92Q mutation of the gene TNFRS1A, which was also present in the affected brother, his daughter and the patient's daughter (Fig. 1) .Both patients began treatment with etanercept, showing a satisfactory initial response. This response was transitory in our index patient, who had to substitute anakinra treatment 3 years after diagnosis due to recurrence of severe exacerbations, while her brother showed a relatively benign course. The descendants of the patients, carriers of the mutation, have not developed symptoms