Selective IgA2 deficiency in a patient with small intestinal Crohn’s disease

The human IgA response is composed of two structurally different subclasses termed IgA1 and IgA2. Compared to IgA1, IgA2 has a shorter hinge region, which makes it more resistant to bacterial proteases. IgA1 is produced both systemically and in mucosal surfaces, whereas IgA2 is mostly confined to the intestines. While the overall IgA response is known to be involved in intestinal homeostasis, the specific contribution of IgA1 and IgA2 remains largely unknown (Chen 2020). Selective IgA deficiency (SIgAD) is the most prevalent primary immune deficiency. About half of SIgAD cases are associated with heterogeneous but generally mild clinical manifestations. Anecdotal evidence of IgA2 deficiency is available (van Loghem 1983, Ozawa 1986, Engstrom 1990), however no associations with clinical manifestations have been reported. Here, we describe the occurrence of a selective IgA2 deficiency in a patient (CD068) with small intestinal Crohn's disease (CD). The patient had undetectable IgA2+ cells and secreted IgA2 antibody in both intestine and circulation. Among other features, patient CD068 presented with duodenal and ileal inflammation. To our knowledge, this is the first case of IgA2 deficiency with a potential link to IBD, which might shed new insights into potential IgA2-specific functions.


FSC-
Interval estimate calculated as 3 X SD around the mean of control HDs.

Tissue immunofluorescence
Immunofluorescence staining was performed on formalin-fixed, paraffin-embedded intestinal tissue sections of 5µm thickness. Samples were deparaffinized through two 5 min xylene baths, with PBST, and secondary anti-IgA2-HRP (Southern Biotech) antibody was added and incubated 1.5h at room temperature. Finally, plates were washed with PBST and TMB substrate was added.
Absorbance was measured at 450nm using a plate reader.

Intestinal biopsy tissue culture
During colonoscopy, biopsies were taken with forceps directly into ice-cold RPMI with 10x Antibiotic-Antimycotic (RPMI-AA) (Gibco), and transported to the lab on ice. Under sterile conditions, biopsies were rinsed on a cell strainer using RPMI-AA 10x. Biopsies were placed in pairs into 48-well plates with 1 ml RPMI-AA 10X per well, and kept from this point at 37°C with 5% CO2. Culture medium was replaced every 2 days using pre-warmed RPMI-AA 2X. Recovered culture medium was centrifuged 10min at 14000G and stored at -80°C.

Bacterial flow cytometry
Fecal pellets were dissolved in PBS at 100 mg/ml by vortexing. Fecal slurry was centrifuged 10min at 50G to remove large particles. Supernatant was filtered through a 40nm cell strainer and cells were then washed by centrifugation at 9000G and resuspension in washing buffer (PBS, 1% BSA, 2mM EDTA). Blocking was done using PBS +1% BSA +20% mouse serum, for 20min at 4°C.
Cells were stained with anti-human IgA2-PE and anti-human IgA-APC for 30min at 4°C, before washing three times with washing buffer. Samples were stained with SYBR green before analysis by flow cytometry.

Genetic Primary Immunodeficiency Panel
A primary immune deficiency panel of 407 genes was obtained with patient's consent (Invitae Corp, San Francisco CA).
Cells were kept in 24-well plates at 37°C with 5% CO2. After 10 days of culture, cells were analyzed by Flow cytometry, as indicated before.

Fecal collection, DNA extraction, and Shotgun metagenomic sequencing.
A stool sample from the index case, CD068 was obtained and compared with stool samples from a cohort of patients with CD and HD. All samples were processed at the Microbiome Bray-Curtis distances). Taxa present in less than 3% of samples, not present above 0.1% relative abundance in at least one sample or annotated as "NA" were filtered out.

Antimicrobial antibody measurement
Patient CD068 plasma was processed at two different time points (2021 and 2023) for specific anti-Saccharomyces cerevisiae antibodies (ASCA IgA and IgG isotypes) as described before (Plevy et al, 2013). The Prometheus Laboratories Biobank of Autoimmune (AI) and Diagnostic Immunology Data Connected to Clinical Phenotypes and Disease Characteristics was interrogated to compare the serological profile from patient CD068 with those from CD patients (n=806), and normal healthy volunteers (NHV) (n=367).

Zonulin measurement
Patient CD068 plasma from two time points (2021 and 2023), as well as plasma from 10 CD patients and 10 Healthy donors were used. Zonulin levels from samples were measured using the Zonulin Serum ELISA (ALPCO) kit following the manufacturer's instructions. Plasma samples were diluted 1:20 and mixed with the biotinylated zonulin family peptide (ZFP) tracer. Samples were aliquoted into the anti-ZFP coated wells in duplicate and incubated for 1 hour at room temperature while shaking. The plate was washed five times with a plate washer and incubated with peroxidase-labelled streptavidin for 1 hour at room temperature while shaking. The plate was washed five times and incubated with tetramethylbenzidine for 15 minutes at room temperature in the dark. The stop solution was added and absorbance was measured at 450nm.

Statistical methods
Unpaired comparisons were done using Mann-Whitney test. When applicable, non-parametric analysis were done using Kruskal-Wallis test and Dunn's multiple comparison test. p values are shown for all comparisons. Interval estimate was calculated as 3*SD around the mean of HD values.