The authors have declared that no competing interests exist.
Kawasaki disease (KD), the leading cause of acquired heart disease in children, primarily affects infants and toddlers. Investigations on immune responses during KD are hampered by a limited understanding of normal immune responses in these ages. It’s well known that Infants have poorer vaccine responses and difficulty with maintaining prolonged serum immunity, but there are few studies on human infants detailing immune deficiencies. Limited studies propose an inability to maintain life-long bone marrow plasma cells. Plasmablasts are a transitional cell form of B cells that lead to long-term Plasma cells. Plasmablasts levels rise in the peripheral blood after exposure to a foreign antigen. In adult studies, these responses are both temporally and functionally well characterized. To date, there have been few studies on plasmablasts in the predominant age range of KD.
Children presenting to an urban pediatric emergency room undergoing laboratory evaluation, who had concern of KD or had fever and symptoms overlapping those of KD, were recruited. Peripheral blood mononuclear cells were isolated and evaluated utilizing flow cytometry with specific B cell markers from 18 KD subjects and 69 febrile controls.
Plasmablast numbers and temporal formation are similar between infectious disease controls and KD subjects. In both groups, infants have diminished plasmablast responses compared to older children.
In this single-time point survey, infants have a blunted peripheral plasmablast response. Overall, similar plasmablast responses in KD and controls support an infectious disease relationship to KD. Future time-course studies of plasmablasts in infants are warranted as this phenomenon may contribute to observed immune responses in this age group.
Kawasaki Disease (KD) is the leading cause of acquired heart disease in children [
KD classically presents as a minimum of five days of fever with conjunctivitis, singular lymph node swelling, oral mucous membrane inflammation, peripheral extremity swelling, and rash [
While the cause of KD is unclear [
During an acute infection, both naïve B cells and memory B cells are stimulated to form plasmablasts: B cells transitioning to plasma cells that circulate in the peripheral blood cell compartment [
Kawasaki disease is most prevelent, from six months to six years of age, at a time when the immune system is still developing. It is known that infants have poorer responses to infections and vaccinations [
This is a single site cross-sectional study of children with fever. Children nine months (chosen for safety considerations) to six years of age presenting to the Emergency Department of Women and Children’s Hospital of Buffalo from March 2014 to May 2016 were screened to determine if they met enrollment criteria. Eligible patients met the following criteria: fever of 38.3°C or above prior to presentation, had a planned blood draw as part of the evaluation of their illness, and one of the following symptoms: rash, mucous membrane changes, extremity changes, conjunctivitis or a single isolated enlarged lymph node. Children admitted or transferred from outside facilities with the specific concern for KD were also enrolled regardless of other criteria. These inclusion criteria captured KD children and a selection of febrile controls. Children with KD in this study are defined as those enrolled who were diagnosed by both primary team and Infectious disease (ID) consultant as having clinical KD and who underwent IVIG treatment after their initial blood draw. Other specific diagnoses were defined on clinical criteria alone, or considered confirmed cases if they included a positive diagnostic test result and chart review revealed clinical illness consistent with the test result. Written informed consent was obtained from parents or legal guardians. Institutional review board (IRB) approval was obtained prior to the initiation of the study.
Initial sampling coincided with admission blood collection and was drawn before IVIG treatment (if applicable). Generally, 5–10 milliliters of blood was drawn up in sodium heparin tubes and placed at room temperature on a neutator in the locked clinical laboratory in the emergency department. Attempts were also made to collect samples 48–72 hours (post- IVIG treatment when applicable) after first blood draw for those who were admitted.
Notable exclusions to prevent effects of excessive blood draws included prior study enrollment within two months, chronic or active blood borne infection (
Generally, we followed established published protocols [
Cells were removed from cryopreservation and thawed in 37°C water bath. Cells were repetitively washed with 10% FBS in PBS. On the final dilution, cells were diluted to 1x106 cells/mL of 2% FBS in PBS. Cells were labeled with Fluorophore-conjugated goat monoclonal antibodies to the following human antigens; CD3, CD14, CD19, CD20, CD27, CD38 and IgG; purchased from Becton Dickinson Bio-sciences (San Jose, CA, U.S.A.) and with IgA+ purchased from Miltenyi (Bergisch Gladbach, Germany). Cells were labeled in 2% FBS/PBS and washed twice with 2% FBS/PBS prior to flow cytometric analysis. Flow cytometric analysis was performed with a FACSAria flow cytometer in a Biosafety Level 2+ laboratory aerosol containment accessory (Becton Dickinson, Franklin Lakes, NJ). Plasmablasts were separated similar to previous published studies [
A single example of the flow gating and analysis is shown. After selecting for single cell events, lymphocyte gate was constructed inclusive of blasting cells, which are enriched for plasmablasts. CD14+ and CD3+ cells were excluded. CD19+/CD20low cells that were dual positive for CD27 and CD38 were defined as plasmablasts.
Methods follow those previously described [
Descriptive characteristics for study subjects were computed after detailed chart review. Categorical variables were reported as proportions in percentage, and continuous level variables were reported as means and standard deviations. Chi-square analysis was used to compare differences in level of white blood cells in urine among KD and control patients. Separate independent t-tests were used to examine differences between patients with Kawasaki Disease and controls for variables that included fever, white blood cell count, platelet count, C-reactive protein (CRP), Sedimentation rate (ESR), hemoglobin, hematocrit, Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), and serum albumin. Flow cytometry data was analyzed using FlowJo software (Ashland, Oregon). Statistical tests, as described in the text, were two-tailed with alpha of 0.05 and performed using Prism software (Graphpad, La Jolla, CA).
Patient ages, gender and discharge diagnosis are listed in
Enrolllee # | Gender | Age months | Febrile days before blood drawn | Clinical Diagnosis and/or associated symptoms and diagnostic result |
---|---|---|---|---|
4 | female | 69 | 7 | Kawasaki Disease |
5 | male | 32 | 7 | Kawasaki Disease, Recurrence of diagnosis 3 months prior |
6 | male | 28 | 6 | Kawasaki Disease, Gallbladder hydrops |
15 | male | 78 | 6 | Kawasaki Disease |
17 | male | 49 | 4 | Kawasaki Disease, Polyarthritis |
24 | male | 58 | 4 | Kawasaki Disease, neck ultrasound with node cluster |
47 | female | 81 | 5 | Kawasaki Disease, + Parainfluenza 2 Respiratory Screen |
53 | male | 29 | 6 | Kawasaki Disease |
67 | male | 10 | 6 | Kawasaki Disease |
68 | male | 21 | 4 | Kawasaki Disease |
75 | male | 20 | 7 | Kawasaki Disease, +Rapid Strep screen, + Echo (lack of tapering, LAD z score 2.5), Gallbladder hydrops, neck ultrasound with node cluster |
79 | male | 26 | 7 | Kawasaki Disease |
80 | female | 53 | 3 | Kawasaki Disease, neck CT with node cluster, retropharyngeal phlegmon |
93 | female | 83 | 5 | Kawasaki Disease, Gallbladder hydrops, neck ultrasound with node cluster |
97 | male | 54 | 6 | Kawasaki Disease, + RSV Respiratory Screen, Gallbladder hydrops |
99 | female | 39 | 9 | Kawasaki Disease |
103 | male | 23 | 5 | Kawasaki Disease |
119 | male | 29 | 6 | Kawasaki Disease |
54 | male | 71 | 14 | Septic Arthritis, pretreated |
66 | male | 40 | 14 | Rash, Conjunctivitis |
94 | female | 42 | 24 | URI, Conjunctivitis (+ Adenovirus) |
123 | male | 30 | 14 | URI, Adenopathy |
45 | female | 72 | 5 | URI, Rash, conjunctivitis |
59 | male | 36 | 6 | URI, Rash, conjunctivitis |
95 | female | 75 | 5 | URI, Rash, conjunctivitis |
131 | male | 19 | 4 | URI, conjunctivitis |
49 | male | 36 | 2 | Rapid Influenza A+, Rash |
101 | female | 85 | 3 | Rapid influenza A + (OSH) |
109 | male | 32 | 2 | Rapid influenza B + (OSH) |
16 | female | 21 | 3 | Hand-foot-and-mouth disease (HFM) |
87 | male | 35 | 5 | Hand-foot-and-mouth disease (HFM) |
104 | male | 28 | 6 | Hand-foot-and-mouth disease (HFM) |
117 | female | 34 | 4 | Hand-foot-and-mouth disease (HFM) |
7 | male | 13 | 4 | MRSA Abscess, Cervical adenitis |
25 | male | 37 | 2 | Cellulitis of the leg |
30 | female | 13 | 3 | Periorbital cellulitis |
42 | male | 15 | 2 | Cervical adenitis; improved on clindamycin |
43 | male | 50 | 4 | Parapharyngeal abscess ( |
48 | male | 14 | 11 | Cervical adenitis; improved on clindamycin |
60 | male | 37 | 7 | Cervical adenitis, conjunctivitis; improved on clindamycin |
98 | male | 14 | 5 | Cervical adenitis, Rash, Conjunctivitis; improved on ampicillin-sulbactam |
107 | male | 88 | 10 | Cervical adenitis; improved on clindamycin |
130 | male | 52 | 7 | Cervical adenitis; improved on clindamycin |
58 | male | 68 | 9 | Pharyngitis, Cervical adenopathy, Rapid strep + |
89 | male | 62 | 4 | Pharyngitis, Rash, Rapid strep + |
92 | female | 20 | 2 | Pharyngitis, Rapid Strep + |
113 | male | 34 | 3 | Pharyngitis, Scarlet fever, Rapid Strep + |
3 | male | 19 | 4 | URI, Rash |
10 | male | 56 | 4 | Viral syndrome, bilateral adenopathy |
12 | male | 27 | 6 | Viral pneumonia |
13 | female | 64 | 3 | Viral pneumonia, Gastroenteritis, Conjunctivitis; + Parainfluenza 3 Respiratory Screen |
14 | male | 22 | 1 | Cough |
20 | female | 14 | 6 | URI, Rash |
29 | female | 52 | 6 | Pyelonephritis, Septicemia |
31 | female | 15 | 5 | Viral syndrome, Neutropenia |
34 | male | 23 | 7 | Bacteremia ( |
52 | male | 80 | 3 | Bacteremia ( |
55 | male | 11 | 7 | Acute pyelonephritis ( |
61 | male | 21 | 5 | Cough, Rash, Conjunctivitis |
64 | female | 82 | 6 | Viral Syndrome, Peeling |
65 | male | 18 | 2 | Viral Syndrome |
69 | female | 27 | 6 | Rash |
70 | male | 9 | 4 | URI |
71 | female | 16 | 3 | Viral syndrome, Rash |
72 | male | 108 | 5 | Viral syndrome, Hepatitis, Rash, Conjunctivitis |
74 | male | 12 | 7 | Viral syndrome, + Enterovirus |
76 | female | 38 | 2 | Allergic reaction with fever, Rash, Hand and feet swelling |
77 | male | 48 | 4 | Viral syndrome, Rash, Allergic drug reaction |
78 | male | 67 | 2 | Viral syndrome, Adenopathy |
81 | male | 11 | 5 | Viral syndrome, Rash |
82 | male | 46 | 2 | URI, Conjunctivitis, Adenopathy |
83 | male | 14 | 6 | Gastroenteritis, Rash |
84 | male | 23 | 4 | URI, Gastroenteritis, Rash |
85 | female | 16 | 1 | Viral Syndrome, Rash |
86 | female | 49 | 4 | Viral syndrome, Gastroenteritis |
88 | male | 43 | 2 | URI |
90 | female | 43 | 6 | Viral syndrome, Gastroenteritis |
91 | female | 57 | 6 | Viral Pharyngitis |
102 | male | 10 | 1 | URI, Cough |
106 | male | 29 | 2 | Erythema multiforme |
110 | male | 59 | 1 | Staph scalded skin syndrome |
112 | male | 11 | 7 | Gastroenteritis |
115 | female | 40 | 4 | Rash, Conjunctivitis |
116 | male | 11 | 6 | Viral syndrome, Rash |
122 | male | 16 | 3 | Rash |
125 | male | 12 | 3 | Rash, Conjunctivitis, referred to rule-out KD |
129 | female | 10 | 1 | URI, Conjunctivitis |
Variable | Kawasaki Disease (KD) |
All Controls |
P-value |
---|---|---|---|
Male Gender, n (%) | 13 (72.2) | 49 (71.0) | 0.920 |
Age in months ±(SD) | 43.4(23.0) | 36.3 (23.6) | 0.955 |
Febrile days prior to blood draw ±(SD) | 5.7 (1.4) | 5.0 (3.7) | 0.208 |
White blood cell count maximum ±(SD) | 17.6 (5.4) | 13.1 (6.0) | |
Platelet count maximum ±(SD) | 393.4 (113.2) | 326.7 (145.9) | |
C-reactive protein [CRP] maximum ±(SD) | 129.4 (62.3) | 73.3 (88.9) | |
Sedimentation rate [ESR], prior to IVIG ±(SD) | 83.6 (30.3) | 55.4 (28.4) | |
Hemoglobin minimum ±(SD) | 10.4 (1.2) | 11.6 (1.0) | |
Hematocrit %, minimum ±(SD) | 31.2 (3.5) | 34.8 (2.9) | |
Aspartate Aminotransferase [AST] maximum ±(SD) | 36.8 (20.0) | 52.4 (31.2) | |
Alanine Aminotransferase [ALT], maximum ±(SD) | 51.2 (36.5) | 57.1 (78.3) | 0.726 |
Serum Albumin minimum ±(SD) | 2.9 (0.5) | 3.7 (0.5) | |
Urine WBC, n (%) |
6 (40) |
22 (78.6) |
Plasmablast levels showed no significant differences between KD and our cohort of febrile controls (medians/means in our KD and control groups were 2.51%/4.53% and 2.32%/4.86% of B cells respectively with ranges in KD patients of 0.31% to 13.45%, and ranges of controls 0.18% to 34.44%) (
Plasmablast levels, as a percentage of overall B cell number, were compared between children with KD (star), prolonged fever (closed circle), and all controls (open circles). Mean values are marked by horizontal dash. Data is shown on a logarithmic scale to improve separation of individual points. Five subsets of all controls that qualified as specific diagnoses are shown (Adenovirus, Influenza, Hand-foot-and-mouth (HFM), Skin and Soft tissue infections (SSTI), and Group A streptococcal pharyngitis (GAS)) and also listed in
Cell Subset | Kawasaki (n = 18) |
Control (n = 69) |
P value | |
---|---|---|---|---|
Percent of Lymphocytes | B cells | 17.3 |
14.8 |
0.081 |
IgG Cells | 1.19 |
0.71 |
||
IgA Cells | 0.69 |
0.56 |
0.237 | |
Plasmablast | 0.33 |
0.29 |
0.607 | |
Percent of B cells | IgG | 7.15 |
4.74 |
0.074 |
IgA | 4.35 |
3.59 |
0.470 | |
Plasmablasts | 2.51 |
2.32 |
0.942 | |
Percent of Plasmablasts | IgG | 11.93 |
9.24 |
0.177 |
IgA | 47.4 |
43.6 |
0.441 |
In KD children, we also noted a similar elevation of B cells consistent with previous publications [
Since plasmablasts can also be highly elevated during autoimmune phenomenon, and levels are shown to correlate with disease flares in these cases [
Linear regression analysis of level of CRP and plasmablast level in KD (starred, small dashed line) and controls (open circles, long dashed line). Results failed to show any linear correlation.
Notably, a number of KD and control children had non-stimulated plasmablast levels, which may be due to a number of factors. First, we evaluated if presentation day of fever of the samples analyzed were different in these subjects. We only had a select few individuals with multiple blood samples, so we chose to pool data by day of fever, similar to previous publications on meningococcal vaccination [
A) Values of means of all samples graphed by day (diamonds) with connected line showing trend. KD samples (stars) and controls (open circles) are shown. B) Showing KD samples alone, single timepoint samples are again labelled with stars. Samples of five individuals with repeat samples are shown by connected (short dash) lines and distinguished by open distinct symbols.
The age of each individual may affect plasmablast levels in direct or indirect fashion. Younger children are more likely to show naïve immune reponses to infection and this has been shown to potentially temporally delay the rise of plasmablasts [
A) In KD children, Linear regression (small dashed line) shows relationship of older children to higher plasmablast levels. B) Mean values for plasmablast percentage of B cells by day of fever for those 24 months and under (long dashed line) compared to subjects over 24 months of age (solid line).
Comparing plasmablast levels in younger children (0–24 months) to older children (25 months and greater; unpaired t-test, Welch’s correction for variable Standard Deviations), indicated that younger children had significantly (p<0.024) lower circulating plasmablast numbers (means and SEM of 2.99 ± 0.63 versus 5.59 ± 0.93; Standard Deviations of 3.62 and 6.87 respectively). Comparing pooled data from days 4–7 (the described peak for a number of viruses in the literature) also shows a significant difference (p <0.007) between these age groups (up to 24 months old n = 20 and greater than 24 months of age n = 33; Mean and SEM of 2.82 ± 0.5931 and 7.097 ± 1.395, and Standard Deviations of 2.65 and 8.02 respectively). This is illustrated by comparing mean results by day in these two age groups (
There are a number of potential immune differences that may explain this. One factor that supports plasmablast formation [
Overall, this work reveals initial observations on overall poor plasmablast response in children at young ages. Generally, it has long been known that infants have a less ‘mature’ immune system than adults. Young children tend to have anti-inflammatory (TH2) skewing of their responses and poor humoral immunity [
B cells are obviously important for adequate response to infectious diseases, but a number of studies support a role for B cell responses in KD as well. Genome wide association studies have identified single polymorphisms in B-cell lymphoid kinas (BLK) and CD40 that correspond to disease risk for KD [
As plasmablasts are enriched for antibodies against the challenging antigen in a number of infections and in vaccinations, we are interested to explore if they can be used to identify the unknown causes of such disorders as Kawasaki disease. For this, our study raises a number of issues. We did observe a variable plasmablast response in KD children which could be seen for a number of reasons. Even on paired sampling, we show a rise in plasmablasts in three of five samples on the second blood draw. Assuming a single etiology sets off KD, potentially, the plasmablast response of the associated infection of KD may have a more unpredictable range than many other infections described [
Considering that it may not be just a single infection, multiple infectious agents with different afebrile prodromes may lead to the KD state. We did not specifically enroll patients in a time course nor recruit on specific days after the beginning of fever, so our data may not overlap with the optimal window to see the plasmablast rise in all patients if this is set off by a variety of infectious agents. Also, viral infections without as much systemic viremia, such as RSV, show overall less responsive and predictable plasmablast peaks [
Two other notable observations are worth mentioning. First, the effect of IVIG on plamasblast responses are unknown. We only have limited data (five paired sample before and after) and with the mixed effects seen (see
This work reveals initial observations on plasmablast responses from natural infections in children at young ages. The similar plasmablast responses shown further support a role for an infectious disease setting off the inflammatory cascade of KD. Future studies evaluating plasmablasts age effects and naïve versus memory responses by repeated sampling over time are warranted to further clarify the normal responses in young children. This will assist in future studies on optimizing vaccine adjuvants in these ages. The similarity of plasmablast responses shown here implies that circulating plasmablasts during KD produce antibodies that specifically target the etiology of Kawasaki disease. Although this might prove difficult for the reasons outlined, further description of the dynamism of these cell levels and studies on the antibodies they express offers the hope of improved diagnostic tests or, potentially, a path to identify the etiology of KD.
This dataset contains all raw values that contributed to the analyses within this manuscript organized by diagnosis. Raw flow cytometry data and data on Interleukin-21 levels are included as well as data and calculations highlighted in Tables
(XLSX)
The authors are thankful to Haiping Qiao for her help with subject recruitment and enrollment. We also acknowledge Jonathon Hoffman, Meghan McLaughlin and Hakimuddin Sojar for their technical support with processing subject samples.