The Serine/Threonine Phosphatase PP4 Is Required for Pro-B Cell Development through Its Promotion of Immunoglobulin VDJ Recombination

PP4 phosphatase regulates a number of crucial processes but the role of PP4 in B cells has never been reported. We generated B cell-specific pp4 knockout mice and have identified an essential role for PP4 in B cell development. Deficiency of PP4 in B lineage cells leads to a strong reduction in pre-B cell numbers, an absence in immature B cells, and a complete loss of mature B cells. In PP4-deficient pro-B cells, immunoglobulin (Ig) DJH recombination is impaired and Ig µ heavy chain expression is greatly decreased. In addition, PP4-deficient pro-B cells show an increase of DNA double-strand breaks at Ig loci. Consistent with their reduced numbers, residual PP4-deficient pre-B cells accumulate in the G1 phase, exhibit excessive DNA damage, and undergo increased apoptosis. Overexpression of transgenic Ig in PP4-deficient mice rescues the defect in B cell development such that the animals have normal numbers of IgM+ B cells. Our study therefore reveals a novel function for PP4 in pro-B cell development through its promotion of VHDJH recombination.


Introduction
B cell development initiates in the bone marrow (BM) of adult mice and is a tightly controlled process. Developing B cells can be divided chronologically into six Hardy fractions (Frs.) A to F according to the recombination status of the immunoglobulin (Ig) heavy chain (HC) locus, the Ig light chain (LC) locus, and the expression pattern of particular cell surface markers [1], [2]. The process starts with D-J H recombination in Fr. A cells, followed by V H -DJ H recombination in Fr. B and Fr. C cells [2], [3]. When a differentiating B cell reaches the Fr. D stage, V L -J L recombination commences [2]. Successful Ig V H DJ H /V L J L recombination leads to the expression of a surface IgM-containing BCR complex that enables a B cell to continue to the Fr. E and Fr. F stages [4]. At the molecular level, DJ H /V H DJ H recombination is initiated when two Ig gene segments flanked by recombination signal sequences (RSSs) are paired and cleaved by RAG [5], [6]. The two gene segments are brought together by the cell's non-homologous end joining (NHEJ) machinery via the sequential recruitment of NHEJ factors. A deficiency of any of these factors results in a failure in DJ H /V H DJ H recombination, an early block in B cell development, and ultimately a shortage of mature B lymphocytes [7].
T cell-specific deletion of PP4 in mice leads to a partial block in thymocyte development at the double negative (DN) stage. The Ca 2+ mobilization and PLC-c1 phosphorylation normally induced by anti-CD3 stimulation are impaired in these PP4-deficient cells [25]. Whether PP4 plays an analogous role in B cell development is unknown. In this study, we utilized mb-1/cre mice to delete the pp4c gene specifically in B cells and identified a pivotal role for PP4 in pro-B cell development. Deletion of PP4 severely disrupted pro-B cell differentiation and consequently led to a complete absence of mature B cells. In PP4-deficient pro-B cells, DJ H recombination was greatly reduced and Ig m HC expression was decreased. We also found that PP4-deficient pre-B cells in vivo accumulated in the G1 phase, showed an elevated level of DNA damage, and underwent increased apoptosis. Significantly, PP4-deficient pro-B cells transgenically expressing IgM successfully differentiated into normal numbers of IgM + B cells. Our results therefore reveal the indispensable role of PP4 in promoting the V H DJ H recombination required for continued pro-B cell differentiation and the production of mature B cells.

Materials and Methods
Mice PP4C F/F mice [25], mb-1/cre mice [31], and Ig HEL transgenic mice [32] generated as previously described were maintained in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Health Research Institutes (NHRI). The Ig HEL transgenic and mb-1/cre mice used in all experiments were heterozygous. All protocols were approved by NHRI's Institutional Animal Care and Use Committee (Permit Number: 099111-A), and all efforts were made to minimize suffering.

BrdU Incorporation in vivo and in vitro
For in vivo BrdU incorporation, mice were injected i.p. with 2 mg BrdU (Simga) in 200 ml sterile PBS at 16 h and 24 h before sacrifice and cell sorting. For in vitro BrdU incorporation, isolated cells were cultured for 16 h in RPMI 1640 medium containing 10% FCS (Hyclone) and 10 mM BrdU prior to analysis using a BrdU-Flow kit (BD Pharmingen TM ).

Comet Assays
To perform comet assays to detect DNA damage, Fr. B and Fr. C cells were sorted by FACSAria (BD) and processed using the OxiSelect TM Comet Assay Kit following the manufacturer's instructions (Cell Biolabs). Data were analyzed using Comet Assay IV TM v4.3 software.

Confocal Imaging
Fr. B and Fr. C cells were sorted, fixed in 4% paraformaldehyde, and permeabilized in 0.2% saponin. After blocking with Image-iT TM FX signal enhancer (Invitrogen), cells were stained with anti-mouse IgM-FITC (HC-specific) antibody (Ab) (BD), seeded on SuperfrostH Plus microscope slides (Thermo Scientific), and mounted using ProlongH Gold anti-fade reagent plus DAPI (Invitrogen).

Ig DJ H Recombination Assay and Coding End Assay
For D H -J H recombination analysis, genomic DNA from 5610 5 cells was extracted and subjected to nested PCR utilizing a primer 59 of the DFL16.1 segment and a primer 39 of the J H 4 segment in the Ig HC locus (Fig. S1). 1 ng of genomic DNA was utilized for first-round PCR using the following primers: dfl16.1:59-CCAGGGCTTTTTGTGAAGGGATCTAC-TACTGT- 39 and jh4:59-TTCTTCAAATGAGCCTC-CAAAGTCC-39. PCR conditions were as follows: 96uC for 3 min; 2 cycles of 94uC for 40 sec, 65uC for 45 sec, and 72uC for 1 min 45 sec; 3 cycles of 94uC, 63uC and 72uC for these same time intervals; and 18 cycles of 94uC, 60uC and 72uC for these same time intervals. For second-round PCR, primers in-dfl16.1:59-AAGGGATCTACTACTGTGTTTATTACTACGGTAG-TAGCTAC-39 and in-jh4:59-AGGCTCTGAGATCCCTAGA-CAG-39 were used with PCR conditions as follows: 94uC for 3 min; 3 cycles of 94uC, 67uC and 72uC for the same time intervals as used for first-round PCR; and 24 cycles of 94uC, 64uC and 72uC for these same time intervals. PCR products were separated by 1% agarose gel electrophoresis and capillarytransferred to nylon membranes. Blots were hybridized with 5 ng/ml DIG-labeled PCR probe synthesized with the following primers: j4p: 59-ATTACTATGCTATGGACTACTGGGGT-CAAGG-39 and in-jh4:59-CAGTAATACATGGCCGTGTCCT-CATACC-39, and detected using the DIG system following the manufacturer's instructions (Roche). To detect DNA doublestrand breaks at Ig loci, oligonucleotides BW-1:59-GCGGTGACCCGGGAGATCTGAATTC-39 and BW-2:59-GAATTCAGATC-39 were annealed for the preparation of BW linker as described previously [33]. 1 mg of genomic DNA from sorted Fr. B cells was treated with 0.1 U/50 ml of Klenow at 25uC for 30 min. After Klenow-inactivation, genomic DNA was washed and ligated with 1000 pM/50 ml of BW linker overnight. 500 ng of ligated DNA was utilized for first-round PCR using the primer 16.1b: 59-GCCTTCCACAAGAGGAGAAG-39 at the upstream of DFL16.1 segment and the primer BW-1 (Fig. S2). PCR conditions were as follows: 96uC for 3 min; 3 cycles of 94uC for 1 min, 58uC for 2 min, and 72uC for 1 min 45 sec; 12 cycles of 94uC for 1 min, 62uC for 2 min, and 72uC for 1 min 45 sec. For second-round PCR, the primer 16.1a: 59-GAAGTCCCCCAGA-GACAGAC-39 at the downstream of primer 16.1b and BM-1 were utilized. PCR conditions were as follows: 94uC for 3 min; 27 cycles of 94uC for 1 min, 62uC for 2 min and 72uC for 1 min. PCR products were separated by 1% agarose gel electrophoresis and capillary-transferred to nylon membranes. Blots were hybridized with 700 ng/ml DIG-labeled PCR probe of 220 bp synthesized with primers 16.1a and COD-R: 59-CCAGGCAG-CACGGTTGAGTTT-39, and detected as described above.

Flow Cytometry
Single-cell suspensions of 1610 6 cells were washed twice with FACS buffer and maintained in the dark at 4uC throughout the experiment. A CantoII flow cytometer (BD) and FACSDiva software (BD) were used for the acquisition of flow cytometric data, and FlowJo software (Tree Star, Inc.) was used for analysis. For apoptosis assays, cells were stained with B220-APC and Annexin V-FITC using the Apoptosis Detection Kit I (BD). To stain and to sort B cells, BM cells were incubated on ice for 15 min with anti-mouse Abs as follows: BP-1-FITC/PE, IgD-Pacific Blue TM (both from Biolegend); IgM-FITC/eFluorH 450 (eBioscience), B220-PE/PerCP/APC-Cy7, CD21-PE, CD5-PE, CD43-APC, CD23-PE-Cy7, CD24-PE-Cy7, and IgM-APC/PE-Cy7 (all from BD). Immunostained cells were washed twice in icecold FACS buffer prior to sorting by FACSAria cell sorter (BD). For intracellular staining, cells were fixed and permeabilized using the BrdU-Flow kit (BD Pharmingen TM ) prior to incubation with m HC-FITC or BrdU-Alexa FluorH 647 (all from BD).

Statistical Analysis
Data were analyzed by a one-tailed distribution, type 3 Student's t-test. Differences between treatment groups with pvalues #0.05 were considered statistically significant.

PP4 Deficiency Leads to a Severe Block in B Cell Development
To study the role of PP4 in B cell development, mb-1/cre mice [31] were bred with pp4c loxp-flanked (floxed) mice [25] to generate mb-1/cre;PP4 F/F (designated here as cKO) mice. As a control, mb-1/cre mice were bred with pp4c WT mice to obtain mb-1/ cre;PP4 +/+ (designated here as WT) mice. B cell subsets, including Fr. E (B220 + CD43 2 IgM + IgD 2 ; immature B cells), and Fr. F (B220 high -CD43 2 IgM low IgD high ; mature B cells), from the BM of WT and cKO mice were analyzed by FACS. In cKO mice, combined staining with anti-B220 and anti-CD43 Abs revealed that the B220 + CD43 2 BM lymphocyte population containing Frs. D to F was greatly reduced (Fig. 1A, left panel). Gating of B220 + CD43 + BM lymphocytes on CD24 plus BP-1 expression showed that the percentage of CD24 + BP-1 + cells (Fr. C) was strongly reduced in the mutant compared to the control (WT, 31.1% vs cKO, 4.7%) (Fig. 1A, middle panel). Among the residual B220 + CD43 2 BM lymphocytes in cKO mice, only IgM 2 IgD 2 cells (Fr. D) were present (Fig. 1A, right panel). Cell count analyses revealed severe reductions in the Fr. C and Fr. D populations in cKO mice, and the complete absence of the Fr. E and Fr. F subsets (Fig. 1B, Table 1). In the spleen and mesenteric lymph nodes (MLN) of cKO mice, mature B cells were barely detectable (Fig. 1C, 1D, Table 2). In addition, CD5 + IgM + B1a B cells were missing from the peritoneum (Fig. 1D). Thus, B cell development in cKO mice is severely blocked before the Fr. C stage, leading to the complete loss of immature and mature B cells.
To determine the deletion efficiency mediated by mb-1/cre, genomic DNA samples from Frs. A-C cells sorted from WT and cKO mice were subjected to PCR analysis. A scheme illustrating the primers used to detect pp4c deletion efficiency is shown in Fig. 2A. In Frs. A-C from cKO mice, pp4c deletion efficiency was 24.4%, 71.4%, and 75.6%, respectively (Fig. 2B, lanes 4-6). In parallel to our deletion efficiency determinations, we extracted total RNA from WT and cKO Frs. A-C cells to analyze PP4 mRNA by RT-PCR (Fig. 2C). After normalization to HPRT expression, PP4 mRNA from cKO mice was reduced by 50% in Fr. B cells and by 77% in Fr. C cells compared to WT controls (Fig. 2D). Thus, consistent with a previous report [34], our results show that mb-1/cre-mediated pp4c gene deletion initiates in Fr. A cells, with higher deletion efficiencies occurring in Fr. B and Fr. C cells. The finding suggests that the developmental block at Fr. C stage in cKO mice likely correlates with the higher pp4c deletion efficiency in this population.

Impaired DJ H Recombination and Increased DNA Doublestrand Breaks at Ig loci in PP4-deficient Pro-B Cells
To further characterize the effects of PP4 deficiency during early B cell development, Frs. A-C cells from WT and cKO mice were subjected to intracellular staining to detect Ig m HC. In WT mice, Ig m + cells constituted 0.3%, 21.8%, and 24.6% of Frs. A, B and C cells, respectively, whereas the corresponding percentages in cKO mice were 0.2%, 6.7%, and 7.6% (Fig. 3A, B). Thus, Ig m HC expression was strongly reduced in the absence of PP4 beginning at the Fr. B stage, an observation confirmed using confocal microscopy to examine Fr. B cells from WT and cKO mice (Fig. 3C). To assess whether this reduction in Ig m HC was due to impaired Ig DJH recombination, we subjected the genomic DNA of WT and cKO Fr. B cells to an in vitro DJH recombination assay. In both WT and cKO Fr. B cells, PCR products representing rearranged DJ H 1, DJ H 2, DJ H 3 and DJ H 4, gene segments appeared when the template was diluted 1:1 (Fig. 3D). In WT mice, the rearranged DJH3 gene segment remained detectable until the template dilution reached 1:16. In contrast, the rearranged DJ H 3 gene segment was detectable up to a dilution of only 1:4 in cKO Fr. B cells, suggesting that PP4 deficiency inhibits D-J H recombination. These data imply that PP4 is involved in the regulation of Ig DJ H /V H DJ H recombination such that loss of pp4c impairs Ig m HC expression.
During VDJ recombination, the hairpin coding ends are generated after the cleavage of recombination signal sequences (RSSs). Before the rejoining process is initiated, hairpin coding ends are opened to form double-strand ends [35]. To address whether the impaired Ig DJ H recombination induced by PP4 deficiency is associated with double-strand breaks (DSBs) at RSSs, the genomic DNA of WT and cKO Fr. B cells was extracted and then subjected to coding end assay as described previously [33], [36]. The PCR product of approximate 350 bp, indicating the DSB at the 39 RSS of DFL16.1 segment (DFL16.1 [39]), appeared in both WT and cKO Fr. B cells (Fig. 3E and Fig. S2). However, the formation of DFL16.1 [39]-coding end in cKO mice was

PP4 Deficiency is Associated with DNA Breaks and Impaired B Cell Homeostasis
During Ig gene rearrangement, DNA breaks are repaired mainly by factors in the NHEJ pathway [37]. The impaired Ig DJ H /V H DJ H recombination by PP4 deficiency raised the possibility that this phenomenon was associated with excessive DNA damage. To address this question, WT and cKO Fr. B and Fr. C cells were subjected to comet assays in which the extent of DNA damage was determined by measuring the percentage of comet tail intensity and the ratio of comet tail length relative to head length. The degree of DNA damage was similar in WT and cKO Fr. B cells (Fig. 4A and Table 3). However, the percentage of tail intensity and the tail length ratio in cKO Fr. C cells were both increased compared to WT Fr. C cell values (Fig. 4A, 4B and Table 3), suggesting that DNA damage is more severe in Fr. C cells lacking PP4.
To understand the effect of PP4 deficiency on cell-cycle progression in vivo, WT and cKO mice were injected with BrdU, and Fr. B and Fr. C cells were isolated and subjected to cell-cycle analysis. Cells of the BrdU 2 7AAD 2 , BrdU + 7AAD 2 , BrdU + 7AAD + , and BrdU 2 7AAD + populations were defined as G1, G1/S, S/G2, and G2/M phase cells, respectively. We found that the percentages of Fr. B cells in each phase were comparable between WT and cKO mice (Fig. 4C). However, in cKO Fr. C cells, a 33% increase in G1 phase cells and a corresponding reduction in cells in the G1/S plus S/G2 phases were observed (Fig. 4C, 4D). To confirm these results in vitro, we cultured WT and cKO Fr. B and Fr. C cells for 16 h in the presence of BrdU and analyzed its incorporation. Similar to our in vivo results, no significant difference in the cell-cycle was observed between WT and cKO Fr. B cells (Fig. 4E). However, compared to WT Fr. C cells, cKO Fr. C cells showed a 2-fold increase in the percentage of G1 phase cells, while the percentages of G1/S and S/G2 phase cells were correspondingly diminished (Fig. 4E). Thus, PP4 deficiency affects the cell-cycle progression of Fr. C cells, leading to an accumulation of G1 phase cells and a reduction in S phase cells. To investigate if PP4 deficiency was associated with increased apoptosis, freshly sorted WT and cKO Fr. B and Fr. C cells were subjected to AnnexinV plus 7AAD staining. While percentages of early apoptotic (AnnexinV + 7AAD 2 ) and late apoptotic (Annex-inV + 7AAD + ) cells were comparable between WT and cKO Fr. B cells, significant increases in early and late apoptotic cells were observed in cKO Fr. C cells compared to WT controls (Fig. 4F,  4G). To confirm this effect on cell viability in vitro, WT and cKO Fr. B and Fr. C cells were cultured in RPMI 1640 medium containing 10% FCS for 16 h followed by 7AAD staining. The percentage of viable cells in the cKO Fr. B culture was drastically reduced compared to the control (WT, 49.4% vs. cKO, 21.7%), as was true for the cKO Fr. C culture (WT, 53.7% vs. cKO, 10.6%) (Fig. 4H). Taken together, these analyses indicate that PP4 deficiency severely interferes with the cell-cycle of Fr. C cells and induces their apoptosis, thereby disrupting B cell homeostasis.

Rescue of the Development of PP4-deficient Pro-B Cells by Ig Transgene Expression
Based on our findings above, we hypothesized that PP4 is required for pro-B cell development because it promotes Ig DJ H / V H DJ H recombination. To test this theory, we crossed our cKO mice to Ig HEL transgenic mice [32] to generate cKO/Ig HEL mice. The Ig HEL transgene drives constitutive BCR expression, which should overcome the developmental block in PP4-deficient pro-B cells if this hurdle is caused by impaired Ig DJ H /V H DJ H recombination [38]. B cell subsets from the BM of cKO/Ig HEL mice were analyzed and compared to those of WT and cKO mice. Interestingly, although numbers of Fr. C and Fr. D cells in cKO/ Ig HEL mice were not restored to WT levels (Fig. 5A, 5B), B220 + IgM + immature B cells and B220 high IgM + B cells became detectable by FACS analysis (Fig. 5A, left panel). By gating on B220 + CD43 2 lymphocytes, we were able to discern that the percentages of IgM + IgD 2 cells (Fr. E) and IgM + IgD + cells (Fr. F) in cKO/Ig HEL mice had increased to 66.7% and 28.8% (Fig. 5A,  right panel), respectively, which were higher than those in cKO mice. Furthermore, cell counts of Fr. E and Fr. F cells in cKO/ Ig HEL mice reached WT levels, indicating that the Ig transgene was able to circumvent the developmental arrest imposed by PP4 deficiency (Fig. 5C). It has been previously reported that Ig transgenic mice show an acceleration in B cell development prior to the Fr. E stage [39]. Thus, the near-absence of Fr. C and Fr. D cells in cKO/Ig HEL mice was likely mediated by their expression of the Ig transgene. We can therefore conclude that the Ig transgene was able to drive PP4-deficient B cell development by promoting the differentiation of IgM + B cells.

Discussion
In this study, we describe the essential role of PP4 in early B cell development through its regulation of Ig DJ H /V H DJ H recombination. Utilizing the mb-1/cre-flox system to generate cKO mice, we have shown that cre activity under the control of the mb-1 promoter initiates at the pre-pro-B cell stage (Fr. A). However, the deletion efficiency of the pp4c allele in Fr. A cells was poor, precluding us from investigating the function of PP4 in this population. Fortunately, acceptable deletion efficiencies were achieved in cKO Fr. B and Fr. C cells, permitting us to determine the effects of PP4 deficiency on these subsets. With respect to Fr. B cells, loss of PP4 leads to impaired Ig DJ H recombination accompanied by reduced m HC expression. These defects occur in the absence of abnormal cell-cycle progression or excessive apoptosis. During WT B cell development, secondary DJ H rearrangements, which employ a more 59 D segment to replace the existing DJ H rearrangement, occur frequently in pro-B and pre-B cells [40]. Hence, it is likely that PP4-deficient Fr. B cells, which fail to complete DJ H /V H DJ H recombination, undergo a secondary DJ H /V H DJ H rearrangement rather than apoptosis. Consistent with that, we found that PP4-deficeint Fr. B cells generate more coding ends at 39 RSS sites of DFL16.1 and DSP. It suggests that the impaired DJ H recombination by PP4 deficiency increases the usage of more 59 D segments for a secondary rearrangement. Very importantly, the analyses of comet scores and apoptosis in Fr. B cells (shown in Fig. 4A and 4F) did not show any significant difference between WT and cKO mice. The data strongly suggest that PP4 regulates Ig DJ H /V H DJ H recombination at the joining phase such that loss of pp4c causes DSBs at Ig loci.
In contrast to Fr. B cells, cells in Fr. C that fail to accomplish a secondary or tertiary DJ H /V H DJ H rearrangement do undergo apoptosis. It has been proposed that such premature death is invoked to prevent the survival of a cell with oncogenic potential [41]. This hypothesis might explain why WT Fr. C cells display more apoptosis than WT Fr. B cells. Even so, apoptosis in cKO Fr. C cells is more severe than in WT Fr. C cells, consistent with the drastic reduction in Fr. C cell counts in the mutant mouse. Because the sole defect in DNA repair during V(D)J recombination would not lead to the formation of visible comets, it is likely that the severe DNA fragmentation in PP4-deficient Fr. C cells is due to other mechanisms, such as apoptosis [42]. Nevertheless, cKO Fr. C cells were observed to accumulate in the G1 phase, indicating a disruption of cell-cycle progression. It is noteworthy that V H DJ H recombination is normally restricted at the G1 phase to ensure the repair of recombination-liberated DNA ends prior to re-entry of the cell-cycle [33]. The accumulation of G1 phase Fr. C cells in cKO mice, together with the strong induction of coding ends-associated DSBs in Fr. B cells, likely reflects the fact that Fr. C cells lacking PP4 cannot complete V H DJ H rearrangement and thus undergo apoptosis. Consequently, PP4 deficiency results in a severe reduction of Fr. C cells and developmental arrest. We were able to restore IgM + BM B cells to WT levels in PP4-deficient mice through transgenic Ig expression. This finding strongly supports our hypothesis that PP4 is indispensable for Ig V H DJ H recombination. It also reveals that the transgenic Ig transmitted functional BCR signaling that overcame the PP4 deficiency and supported the differentiation of IgM + B cells.
Intriguingly, the phenotype of mice bearing a B cell-specific deletion of PP4 is more drastic than the block in T cell development exhibited by mice bearing a Lck-cre-mediated T cell-specific deletion of PP4, as CD4 + and CD8 + single positive T cells are present in both the thymus and periphery of these mutants [25]. During T cell development, DN2 and DN3 thymocytes, which are undergoing TCRß gene rearrangement, are the cells equivalent to the developing Fr. B cells. Because the deletion efficiencies in DN2 and DN3 thymocytes from those mutant mice were not reported, it was not clear whether creaffiliated leakiness could result in a less severe development block  than the effect of mb-1/cre mediated PP4 deficiency in B cells. Although the mechanism for VDJ recombination is in general highly conserved between B and T lymphocytes, gene-targeting of NHEJ factors in some cases can lead to unequal severities in B and T cell developments. For example, in Ku70 knockout (KO) mice and DNA polymerase m (Pol m) KO mice, VDJ recombination at Ig loci is drastically impaired, whereas TCRß/a gene rearrangement is functional [43] [44] [45]. A comprehensive recombination assay in PP4-deficient thymocytes would be necessary to address whether PP4 is also involved in TCR gene rearrangement. PP4 has been implicated in two DNA repair pathways: homologous recombination-mediated repair of DNA breaks via dephosphorylation of cH2AX [18], [24], and NHEJ-mediated end-joining by dephosphorylation of KAP1 (KRAB-associated protein 1) [46]. Whether the disruption of cH2AX or KAP1 phosphorylation status can lead to abrupt developmental arrest at the pro-B cell stage requires further investigation. In any case, the results of our study provide the first genetic evidence establishing a critical role for PP4 in pro-B cell development through its promotion of Ig V H DJ H recombination.