Homeodomain-interacting protein kinase (HIPK)-1 is required for splenic B cell homeostasis and optimal T-independent type 2 humoral response

Abstract

The homeodomain-interacting protein kinase (HIPK) family is comprised of four highly related serine/threonine kinases originally identified as co-repressors for various homeodomain-containing transcription factors. The HIPKs have been shown to be involved in growth regulation and apoptosis, with numerous studies highlighting HIPK regulation of the tumor suppressor p53. In this study, we have discovered a B cell homeostatic defect in HIPK1-deficient (HIPK1(-/-)) mice. Lymphopoietic populations within the thymus and bone marrow of HIPK1(-/-) mice appeared normal based upon FACS analysis; however, the spleen exhibited a reduced number of total B cells with a significant loss of transitional-1 and follicular B cell populations. Interestingly, the marginal zone B cell population was expanded in HIPK1(-/-) mice, yielding an increased frequency of these cells. HIPK1(-/-) B cells exhibited impaired cell division in response to B cell receptor cross-linking in vitro based upon thymidine incorporation or CFSE dilution; however, the addition of CD40L rescued HIPK1(-/-) proliferation to wild-type levels. Despite the expanded MZ B cell population in the HIPK1(-/-) mice, the T-independent type 2 humoral response was impaired. These data identify HIPK1 as a novel kinase required for optimal B cell function in mice.

Figure 1. HIPK1 is expressed in hematopoietic cells.

A, Analysis of the tissue-specific gene expression profiles of hipk1 and hipk2 using the BioGPS-Gene Portal Hub web application (Genomics Institute of the Novartis Research Foundation (GNF). For this study, B and T lymphocytes were obtained from spleen. B, Primary B and T lymphocytes were isolated from the spleens of HIPK1^+/+ (WT) and HIPK1^−/− (KO) mice. mRNA was isolated and reverse transcribed to cDNA. PCR amplification was used to determine if hipk1 was expressed. gapdh was used as a control.

Figure 2. HIPK1^−/− mice exhibit normal T cell development in thymi.

A, Thymic cellularity was similar between HIPK1^+/+ and HIPK1^−/− mice at 4–5 weeks and 8–13 weeks. For 4–5 week old mice, n = 7; for 8–13 week old mice, n = 13. B, CD4 and CD8 expression was used to identify the frequency of double-positive (DP) and single positive populations, and did not reveal any abnormalities. FACS analysis of thymocyte subsets using CD44 and CD25 expression indicated that the double-negative DN I–IV populations were intact in HIPK1^−/− mice. Representative FACS plots are shown (n = 9).

Figure 3. HIPK1^−/− mice exhibit normal B cell development in the bone marrow.

A, FACS analysis of bone marrow. Bone marrow was isolated, ACK-treated and stained with B220, IgM, BP.1, CD43, HSA and IgD in order to determine the frequencies of developing B cell subsets; representative FACS plots are shown (n = 8).

Figure 4. Analysis of spleens from HIPK1^−/− mice.

A, Mice were weighed after being sacrificed. Spleens were then excised and weighed, and expressed as a percentage of the total mouse weight (n = 16). B, The number of splenocytes obtained after ACK-treatment was counted using a hemacytometer (n = 7). C–D, FACS was used to identify splenic populations. Representative FACS plots are shown; stains were performed in at least three independent experiments with similar results unless indicated otherwise. C, CD3 was used to identify T cells, and CD19 to identify B cells (n = 7). D, CD4 and CD8 were used to identify splenic T cell subpopulations (n = 6). E, CD11b and F480 were used to identify macrophages. CD11c, CD4 and CD8 were used to identify dendritic cells (n = 3). *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 5. Splenic B cell homeostasis is disrupted in HIPK1^−/− mice.

A, Splenic B cells were stained using CD23, CD21, and IgM to distinguish between B cell subsets. Representative FACS plots are shown for experiments that were conducted at least three times with similar results. B–D, Frequencies obtained by FACS were converted to absolute numbers and averaged. B, The absolute number of T1 B cells was reduced in HIPK1^−/− mice, whereas the absolute number of T2 B cells was unaffected (n = 6). C, The absolute number of MZ B cells was elevated in HIPK1^−/− mice (n = 6). D, The absolute number of FO B cells was reduced in HIPK1^−/− mice (n = 7). E, The MZ B cell population expressed as a percentage of the total B cell population. F, Cells obtained from peritoneal lavage were stained with CD5 and IgM to identify the B1 B cell population by FACS (n = 6). *p≤0.05, **p≤0.01.

Figure 6. Enlarged MZ compartment in HIPK1^−/− mice.

Splenic sections were stained with IgM (blue) and IgD (red) or MAdCAM1 (red) to identify the MZ B cell population and the marginal sinus. Representative sections are shown, with similar results observed in three mice.

Figure 7. Basal serum Ig levels.

A, Basal serum Ig levels from 12 week-old HIPK1^+/+ and HIPK1^−/− mice were measured by ELISA. n = 4, except for IgG2c, where n = 8. *p≤0.05, **p≤0.01.

Figure 8. HIPK1 is required for BCR-induced proliferation.

A, Proliferation of HIPK1^+/+ and HIPK1^−/− splenic B cells in response to anti-IgM ± CD40L (10 µg/ml each) was assessed by [3H]-thymidine incorporation. Cultures were pulsed with 1 µCi tritiated thymidine 12 hrs before the indicated time points. Representative results of three independent experiments are shown. B, Cell division of HIPK1^+/+ and HIPK1^−/− splenic B cells was determined by CFSE dilution assay. Cells were stimulated with anti-IgM ± CD40L (10 ug/ml) or media alone, and analyzed at 24, 48, and 72 hrs post-stimulation. The solid peaks are wild type and the empty peaks are HIPK1^−/−. FACS plots are representative of three independent experiments. C, Viability was measured by FACS by gating on the AnnexinV and PI double-negative populations at 48 hrs after stimulation (n = 4). *p≤0.05.

Figure 9. HIPK1^−/− mice exhibit an impaired TI-2 humoural response.

A, The TI-2 response was evaluated by measuring TNP-specific IgM and IgG3 serum levels after i.p. immunization with 25 µg TNP-Ficoll. Mice were bled at the indicated time points and serum antibody isotypes were measured by ELISA, (n = 5). B, Elispot analysis of TNP-specific-IgM-producing cells in response to TNP-Ficoll seven days post i.p. immunization (n = 4). *p≤0.05, **p≤0.01, ***p≤0.001.

Figure 10. BCR signaling in the absence of HIPK1.

A, Primary B cells were stimulated with anti-IgM (10 µg/ml) for the indicated amounts of time and then lysed. Lysates were subjected to SDS-PAGE and were probed with phospho-specific antibodies as indicated. Blots are representative of minimally three independent experiments.