3.1. Frequency of ILCs in HSP patients in the peripheral blood
In this study, 51 HSP patients and 22 normal controls were included. There were no differences in age and gender between the two groups. Selected laboratory test results and different subtypes of HSP patients are presented in Table 1.
According to the gating strategy as shown by Sandra Bonne-Année et al [15], the isolated PBMCs from HSP patients and normal controls were stained, and then they were subjected to flow cytometry for the analysis of ILC subsets. The lineage makers (CD3, CD45, CD19, CD14, CD1a, CD94 and CD34) are used to exclude B lymphocytes, T lymphocytes, monocytes, dendritic cells, NK cells and hematopoietic stem cells. Lin-CD127 + cells gated on CD45 + lymphocytes were considered to be ILCs (Fig. 1A–C). To further differentiate subtypes, ILCs expressing CRTH2 + were defined as ILC2 (Fig. 1B and C). CD117- CRTH2- ILCs were designated as ILC1 and CD117 + CRTH2- ILCs were passed as ILC3(Fig. 1). As shown in Fig. 1B from a typical HSP individual, ILC1, ILC2 and ILC3 could be differentiated clearly.
3.2. ILC subsets were altered in HSP patients
In HSP, ILC1 were significantly increased and ILC3 were significantly decreased (Fig. 2). The difference of ILCs/Lymphocytes and ILCs/PBMC between the HSP group and the normal controls were statistically significant (P = 0.036,0.026 respectively). There were significant increases in ILC1/ILCs and decreases in ILC3/ILCs, however the percentage of ILC2/ILCs in patients with HSP was not significantly different from that of the normal control group(P > 0.05). In comparison to normal controls, the difference of ILC1/ILC3 was statistically significant (P < 0.001), in other words, the ratio of ILC1/ILC3 was significantly higher in HSP patients, nevertheless, there were no difference in ILC1/ILC2 between the two groups(P > 0.05).
3.3. The ratio of ILC1/ILCs and ILC3/ILCs in HSP patients restored after treatment
Sixteen patients were reexamined after the initiation treatment with methylprednisolone for about 7 to 10 days. There were no changes in ILCs/Lymphocytes and ILCs/PBMC before and after treatment (P = 0.833,0.940 respectively). But there were changes in ILCs subsets. As shown in Fig. 4A–C, ILC1 were significantly decreased(P < 0.001) while ILC3 increased(P = 0.033), there was no significant change in ILC2(P = 0.143). In addition, the difference of ILC1/ILC3 was statistically significant before and after treatment in HSP patients(P < 0.001), namely, ILC1/ILC3 was significantly decreased after treatment, while no difference in the ratio of ILC1/ILC2 before and after treatment(P = 0.460) (see Fig. 3).
3.4 ILC subsets in different subtypes of HSP patients
According to the clinical manifestation, the patients of HSP are divided into five subtypes: purpura type, arthritis type, abdominal type, renal type and mixed type. In contrast to normal groups, the ratio of ILCs/Lymphocytes increased in patients of arthritis type and mixed type (P = 0.014,0.039 respectively), ILCs/PBMC also increased in the same two subtypes(P = 0.010,0.034). There was no difference in ILCs/Lymphocytes and ILCs/PBMC among patients of purpura type, abdominal type, renal type in compare with normal controls (P > 0.05). ILC1 accounted for a relatively high proportion in purpura type, arthritis type, abdominal type and mixed type except for renal type (P = 0.027,0.007, P < 0.001, P < 0.001 and P > 0.05). ILC3 decreased in abdominal type and mixed type, and the difference was statically significant(P = 0.015,0.006). Furthermore, ILC1/ILC3 in all subtypes of HSP was higher than that of the normal control group, and the difference was statistically significant(P < 0.05) (see Fig. 4).
3.5 CD8 + T cell was positively correlated with ILC1/ILCs
HSP is a kind of leukocytoclastic vasculitis that is characterized by an immune complex mediated type III allergic disease. Dominant IgA deposits in the affected capillaries and small blood vessels. Besides IgA, IgG, IgM and T lymphocytes as well as other immune cells, cytokines and complement also participate in the pathogenesis of HSP [16]. Namely, there are cell-mediated immunity and humoral immunity disorders in HSP. Serum IgA increased, and IgA deposition was observed in kidney and skin mucosa. The levels of CD19 + CD23 + and CD3 + CD4+/CD3 + CD8 + were decreased. ILC1/ILCs was positively correlated with CD3 + CD8 + T lymphocytes (r = 0.3701, p = 0.0075), however, there were no significant correlations between ILC3/ILCs (r=-0.2747, p = 0.0511), ILC1/ILC3 (r=-0.2669, p = 0.0583) and CD3 + CD8+.
The level of IgA did not correlate with ILC1/ILCs (r = 0.0242, p = 0.8660), ILC3/ILCs (r = 0.0358, p = 0.8033) and ILC1/ILC3 (r = 0.0604, p = 0.6739). There were also no correlations between CD19 + CD23 + and ILC1/ILCs (r=-0.1826, p = 0.1998), ILC3/ILCs (r = 0.0881, p = 0.5386) and ILC1/ILC3(r=-0.1036, p = 0.4693). CD3 + CD4+/CD3 + CD8 + did not correlate with ILC1/ILCs (r=-0.0676, p = 0.6407), ILC3/ILCs (r = 0.0143, p = 0.9213) and ILC1/ILC3 (r=-0.0201, p = 0.8899), too. The relationship between CD3 + CD4 + and ILC1/ILCs (r = 0.0400, p = 0.7803), ILC3/ILCs (r=-0.0426, p = 0.7668) and ILC1/ILC3 (r = 0.0629, p = 0.6608) was not significant (see Fig. 5).