Characterization of adipose-derived stromal/stem cells from the twitcher mouse model of krabbe disease

Xiujuan Zhang
Julie A Semon
Shijia Zhang
Amy L Strong
Brittni A Scruggs
Jeffrey M Gimble
Bruce A Bunnell
Characterization of adipose-derived stromal/stem cells from the twitcher mouse model of krabbe disease
R E S E A R C H A R T I C L E Open Access
Background: Krabbe disease, also known as globoid cell leukodystrophy, is an autosomal recessive neurodegenerative disease caused by the genetic deficiency of galactocerebrosidase (GALC), a lysosomal enzyme responsible for the degradation of several glycosphingolipids like psychosine and galactosylceramide. In order to investigate whether GALC deficiency in Krabbe disease affects adipose-derived stromal/stem cell (ASC) properties and if the ASCs could be used as a source of autologous stem cell therapy for patients with Krabbe disease, ASCs isolated from subcutaneous adipose tissue of Twitcher mice (a murine model of Krabbe disease) and their normal wild type littermates were cultured, expanded, and characterized for their cell morphology, surface antigen expression, osteogenic and adipogenic differentiation, colony forming units, growth kinetics, and immune regulatory capacities in vitro. Results: ASCs from Twitcher mice (TwiASCs), when compared to ASCs from normal mice (WtASCs), have a reduced osteogenic differentiation potential, have less self-replicating and proliferative capacity, although they have the same fibroblast morphologies and cell sizes. However, surprisingly, the TwiASCs demonstrated similar immune-suppressive capacities as their counterparts WtASCs did when they were transwell co-cultured with macrophages in vitro.Conclusion: This study reveals that Twitcher ASCs exhibit differences in the biologic potential when compared to their counterparts from normal mice. The changes in Twitcher ASCs may be influenced by the GALC deficiency in Twitcher mice. Nevertheless, none of the changes preclude the use of the TwiASCs for autologous applications.

Background

Krabbe disease, also known as globoid cell leukodystrophy, is an autosomal recessive neurodegenerative lysosomal storage disorder affecting both the central nervous system (CNS) and peripheral nervous system (PNS) [1]. The disease is caused by the genetic deficiency of galactocerebrosidase (GALC), a lysosomal enzyme responsible for the degradation of several glycosphingolipids like psychosine and galactosylceramide [1][2][3][4]. The accumulation of GALC substrates, especially psychosine, leads to progressive demyelination of axons and death of oligodendrocytes in the CNS and Schwann cells in the PNS [5].

Based on the age of onset and severity, Krabbe disease is clinically divided into infantile type, juvenile type and adult type [6]. The infantile Krabbe disease is most common with onset between 3-6 months of age of symptoms including vomiting, difficulty in feeding, extreme irritability, complete blindness, deafness, spastic paralysis, extreme emaciation and dementia, leading to death by 2 years of age [6][7][8][9][10]. Currently, there is no cure for Krabbe disease. The only approved clinical therapies for Krabbe disease are bone marrow transplantation (BMT) and umbilical cord blood transplantation (UCB), after myeloablative chemotherapy, and such a procedure has only been beneficial in the infantile type if it was performed before the onset of clinical symptoms [7,11]. However, the majority of pre-symptomatic infants receiving transplantation have been reported to develop severe disabilities, including motor and language deterioration [11][12][13]. Moreover, BMT and UCB therapies are limited by the scarcity of donors, and can be complicated by the development of graft versus host disease (GVHD), a serious and often fatal condition resulting from the immunological attack of foreign T cells against the patient [14]. One study had reported that GVHD developed in 8 out of 11 asymptomatic and 5 out of 14 symptomatic Krabbe disease patients treated with UCB [11].

Adipose-derived stromal/stem cells (ASCs) can be isolated from lipoaspirate following liposuction. ASCs have the potential to differentiate into adipocytes, osteocytes, chondrocytes, myocytes and neuronal cells when stimulated with appropriate induction factors [15][16][17][18][19][20][21]. ASCs have received increasing attention from both biological scientists and clinicians because of the cells' ability to differentiate into multiple lineages, the abundance and easy accessibility of adipose tissues, and the feasibility of harvest by a minimally invasive procedure. In addition, ASCs have been reported to reduce inflammation [22,23] and secrete growth, angiogenic and anti-apoptotic factors [24,25]. Therefore, ASCs from individual Krabbe disease patients may be a source of mesenchymal stromal/stem cells for autologous cell therapy. Autologous transplantation has lower rates of post-transplant complications and infections compared to allogeneic transplantation. Moreover, there are no concerns about donor availability and risk of histoincompatibility in autologous transplantation. Autologous ASCs offer numerous advantages from regulatory, histocompatibility, and immunological perspectives [26]. The Twitcher mouse model was developed through spontaneous mutation of the GALC gene at the Jackson Laboratory in 1976 [8,27,28] and shares many neuropathologic findings with the human infantile type of Krabbe disease. In this study, ASCs harvested from Twitcher mice (TwiASCs) and normal wild type mice (WtASCs) from the same litters were culture expanded, characterized, and compared based on features such as differentiation potential, colony forming unit capabilities, cell surface marker profiles, growth kinetics and cytokine expression.

Results

Morphology and cell size of TwiASCs and WtASCs

The cell morphology of TwiASCs was consistent with that of the WtASCs, which were fibroblast-like in appearance ( Figure 1A). The sizes of both cell types were also analyzed based on the forward scatter signals of flow cytometry. As shown in Figure 1B, the cell size of TwiASCs versus WtASCs was not significantly different (t-test, P>0.05).

Flow cytometric analysis of surface antigens on TwiASCs and WtASCs

ASCs from Twitcher mice and normal wild type mice were isolated, cultured, expanded, and analyzed for surface markers by flow cytometry at passage 3. ASC surface markers CD29, Sca1, and CD106; hematopoietic markers CD34 and CD45; phagocytic lineage marker CD11b; and endothelial marker CD31 were tested. As shown in Figure 2, both TwiASCs and WtASCs were positive for CD29, Sca1, and CD106. Both cell types are negative for CD45, CD11b, and CD31. However, there is a significant difference in CD34 expression between TwiASCs and WtASCs (t-test, P<0.05). The ASCs were harvested from three Twitcher mice and three wild type mice respectively, and both TwiASCs and WtASCs were repeated three times independently by flow cytometry and similar results were obtained in all studies.

Differentiation assays for TwiASCs and WtASCs

Twitcher ASCs and wild type ASCs were cultured in osteogenic and adipogenic differentiation medium for 3 weeks to test their lineage differentiation efficiency. On day 21, the ASCs were stained with Alizarin Red to assess bone mineralization, and with fresh Oil Red O for lipid droplets. Both TwiASCs and WtASCs efficiently differentiated into osteocytes and adipocytes ( Figure 3). However, as shown in Figure 3A, the WtASCs appear to have greater differentiation capacity for osteogenic differentiation than the TwiASCs. Quantification of the differentiation levels of both cell types ( Figure 3B) further demonstrated that WtASCs (OD ratio=11.39±0.73) could differentiate into osteocytes to a much greater degree than TwiASCs (OD ratio=5.78±0.17) (t-test, P<0.05) over the period of the assay. Quantification of adipocyte differentiation indicated that WtASCs (OD ra-tio=1.20±0.07) have a slightly higher capacity than TwiASCs (OD ratio=1.01±0.10), but not statistically significant (t-test, P>0.05). The graph in Figure 3B represents the ratios of OD of differentiated cells normalized to control cells.

Osteogenic markers in WtASC and TwiASC

As shown in Figure 3, WtASCs appear to have greater differentiation capacity for osteogenic differentiation than the TwiASCs. To further investigate their osteogenic potentials, replicate cultures of WtASC and TwiASC were induced to undergo osteogenic differentiation and harvested on day 7, 14 and 21 for alkaline phosphatase (ALP), runt-related transcription factor 2 (RUNX), and osteocalcin (OCN) mRNA analysis by real-time PCR. ALP catalyzes the hydrolysis of monophosphate esters at a high pH, and it is one of the first functional genes expressed in the process of calcification [29]. RUNX is a bone-specific transcription factor, and it stimulates OCN and other bone specific gene transcriptions via the binding of its runt domain to their promoters [30]. OCN, a marker of bone formation, is the most abundant and most widely studied non-collagenous protein in bone [31]. As demonstrated in Figure 4, WtASCs have a much higher ALP mRNA expression than TwiASC on day 7 (t-test, P<0.05). By day 21, WtASCs demonstrated higher expression in all the three lineage markers than TwiASC (t-test, P<0.05) (Figure 4).

Colony forming unit assay

The colony forming unit (CFU) assay was used to assess the self-renewal ability of the cells. Both TwiASCs and WtASCs were seeded onto 56.7 cm 2 Nunc cell culture plates at a total of 100 cells per plate and the number of colonies was enumerated after 14 days of culture. The CFU assay results ( Figure 5) demonstrated that WtASCs have a significantly higher self-replicating capacity (6.20±1.62) than TwiASCs (1.60±0.51) (t-test, P<0.05).

Growth kinetics of TwiASCs and WtASCs

In order to test their proliferative capacity, TwiASCs and WtASCs were plated on 56.7 cm 2 Nunc cell culture plates at a density of 500 cells/cm 2 and cultured for 10 days. The fold-increase in cell number was analyzed every 2 days throughout the culture period and was calculated by comparing the cell number at each time point to the original plating numbers. As illustrated in Figure 6, the TwiASCs have a much slower growth rate than WtASCs. For the 10-day culture period, TwiASCs only had a maximal foldincrease highest of 5.49 ±1.29 while WtASCs had a maximum of 12.10 ±0.38. After that, both TwiASCs and WtASCs reached their growth plateau and started to decline.

Regulation of TwiASCs and WtASCs on inflammatory cytokine expression in primary macrophages

The thioglycolate-elicited peritoneal primary macrophages were seeded onto 24-well cell culture plates. After 6-hour incubation, 0.4 μm pore size transwells were placed in the 24 well plates and TwiASCs and WtASCs were added on top of each transwell. The transwell coculture was incubated overnight followed by stimulation of the cells with LPS for an additional 24 hours. The expression levels of inflammatory cytokines, IL-1α, IL-1β, TNFα, IL-6 and IL-10 were analyzed by real-time PCR to determine whether TwiASCs and WtASCs could effectively regulate their expression. As illustrated in Figure 7, little cytokine production was observed in macrophages in the absence of LPS, and LPS stimulation triggered an outburst of cytokine production by macrophages, especially the pro-inflammatory IL-1α, IL-1β, TNFα, IL-6 cytokines. Remarkably, WtASC significantly reduced the macrophage ability to produce
IL-1α (t-test, P<0.01), IL-1β (t-test, P<0.01), TNFα (t-test, P<0
.01), IL-6 (t-test, P<0.05), and enhanced the production of the anti-inflammatory cytokine IL-10 (t-test, P<0.05). TwiASCs demonstrated similar immunesuppressive capacities in vitro as WtASCs with the exception that they did not suppress the IL-6 expression by the macrophages (Figure 7, t-test, P>0.05).

Discussion

This study compared adipose stem cells from Twitcher mice to those isolated from their wild type littermates in order to determine whether ASCs with the mutation associated with Krabbe disease continue to display normal phenotype. This has relevance to their potential use in future transplantation studies.

From the morphologic perspective, TwiASCs and WtASCs appeared similar with a fibroblast-like morphology and had no significant difference in cell size based on direct observation and on the forward scatter data of flow cyotmetry. Both TwiASCs and WtASCs were analyzed by flow cytometry for specific cell surface markers such as CD29, CD106, Sca1, CD45, CD34, CD11b, and CD31. The cell surface marker profiles for both cell types were comparable with the exception of CD34 expression where there was a significant difference between TwiASCs and WtASCs. The expression of CD34 differed as a function of passage numbers for TwiASCs. It was positive in passage 3 as shown in Figure 2, but gradually diminished its expression along higher passages, which is typical of ASCs in culture (data not shown). There have been reports showing that CD34 is consistently down-regulated while culturing over several passages [24,32], which is consistent with our findings that TwiASCs gradually lost their expression of CD34 over progressive passages while all other surface markers tested remained the same. However, there have been never detectable levels of CD34 expression in WtASCs. CD34 is a surface marker for hematopoietic stem cells, endothelial progenitor cells and satellite cells. The difference of CD34 expression between TwiASCs and WtASCs may be due to differences between the adipocyte niches within adipose depots of Twitcher mice and their wild type littermates.

Both TwiASCs and WtASCs could be readily differentiated into osteocytes and adipocytes. Although the lipid droplets produced by WtASCs were larger, in general, than those of TwiASCs as observed under the microscope, there was no significant difference in the accumulation of neutral lipids between TwiASCs and WtASC. However, WtASCs exhibited an increased differentiation along the osteogenic lineage relative to TwiASCs. The study of osteogenic markers in differentiated WtASC and TwiASC (Figure 4) demonstrated that WtASCs, compared to TwiASCs, have significantly increased levels of expression. ALP is a cell surface glycoprotein that is involved with mineralization and its role in mineralization process occurs during early differentiation. Measurement of increased ALP expression at the mRNA level has been taken as a reliable indication of the osteoblastic phenotype [29,33]. OCN is a matrix protein that regulates osteoclast activity, and is a late marker of differentiation at the mineralization stage [33]. Reduced osteogenic differentiation capacity of TwiASCs correlated with reduced ALP and OCN expression of TwiASCs, although the mechanism(s) regulating the reduced ALP and OCN expression in TwiASCs is unclear. However, it has been noticed that Twitcher mice demonstrated postnatal bone retardation, and Contreras and colleagues [34]  Twitcher mice demonstrated osteopenic phenotype, and their bones were much smaller and weighed less compared to normal mice. The same group [34] also reported that the accumulated psychosine induced by GALC-deficiency is one of the potential factors that resulted in the stunted bone growth in Twitcher mice. Psychosine accumulation in TwiASCs may play a role in their reduced differentiation capacity along the osteogenic lineage.

The colony forming unit assay and growth curve analyses were used to compare the self-renewal and proliferative capacities between the TwiASCs and WtASCs. The results clearly indicated that the TwiASCs have a significantly reduced ability to form single-cell-derived colonies when plated at low density in comparison with the WtASCs. Moreover, cultures of TwiASCs seemed to grow much slower than did their wild type counterparts. However, many studies have demonstrated that the therapeutic effects of ASCs are not only solely the results of their capacity for differentiation along mesenchymal and non-mesenchymal lineages, but more importantly related to their immune regulatory capacities [23,[35][36][37][38]. Macrophages are a key component of the innate immune system and are widely distributed in many different tissues. In addition to their role in initial phases of immune defense, macrophages play an indispensable role in later phases of tissue homeostasis and repair. The interaction of mesenchymal stem cells (MSCs) with macrophages has been widely investigated in recent years [39][40][41][42]. Ne'meth et al. [40] demonstrated that MSCs increased the IL-10 production by LPS-stimulated macrophages in vitro. They also demonstrated that administration of # * * * Figure 4 Real-time PCR analysis of osteogenic lineage marker expression in differentiated TwiASCs and WtASCs. TwiASCs and WtASCs were induced for osteogenic differentiation. On day 7, 14 and 21, the cells were harvested and total RNA was extracted for PCR analysis of osteogenic markers ALP, RUNX and OCN. # means P< 0.05 vs WtASCs on day 7 and * means P< 0.05 vs WtASCs on day 21 for ALP, RUNX and OCN respectively (t-test, n=6). # Figure 5 Colony forming unit assays for TwiASCs and WtASCs. 100 cells were plated on 56.7 cm 2 Nunc cell culture plates and incubated for 14 days. Cells were stained with 3% crystal violet, and colonies 2 mm or larger in diameter were counted. # means P< 0.05 vs WtASCs (t-test, n=5).

MSCs improved organ function and reduced mice mortality for the treatment of sepsis, and the therapeutic effects of MSCs were induced upon their stimulation by IL-10 produced by macrophages. In our study, the TwiASCs and WtASCs were co-cultured in transwell plates with peritoneal macrophages stimulated by LPS, and the in vitro expression of inflammatory cytokines by macrophages was analyzed by real-time PCR. The results indicated that TwiASCs demonstrated similar immunesuppressive capacities in vitro as their counterparts WtASCs. These data suggested that, although TwiASCs had reduced osteogenic differentiation, self-renewal and proliferative capacities, they are quite similar to WtASCs in their anti-inflammatory properties. This is an important finding since the therapeutic effects of adult stem cell therapy had been shown more related to their immunemodulatory capacities.

Conclusion

ASCs from Twitcher mice, when compared to ASCs from normal mice, have a reduced osteogenic differentiation potential, have less self-replicating and proliferative capacity, although they have the same fibroblast morphology and cell sizes. However, surprisingly, the TwiASCs demonstrated similar immune-suppressive capacities as their counterparts WtASCs did when they were cocultured with macrophages in vitro. These findings suggest that while the GALC deficiency in Twitcher mice does influence ASC profiles in some aspects, it does not affect their immune-suppressive capacities significantly. These outcomes have relevance since the immune-suppressive function is potentially a critical mechanism that would account for the therapeutic benefit of ASC transplantation in Krabbe disease.

Methods

Isolation and culture of adipose-derived stem cells

The Twitcher mice and their littermates were obtained from breeding pairs carrying a natural mutation in the GALC gene. Breeding pairs were originally purchased from the Jackson Laboratory (Bar Harbor, ME). All the protocols and experimental procedures were approved by the Institutional Animal Care and Use Committee at Tulane University. Genotyping of Twitcher mice was performed on post-natal day 14 by real time PCR modified as previous described [43]. The subcutaneous fat pads were isolated from the post-natal day 34 moribund Twitcher mice and their wild type littermates, rinsed with Hank's balanced salt solution (HBSS) (Life Technologies, Grand Island, NY) to remove blood and hair contamination, and digested with 0.1% collagenase type 1 solution (Life Technologies) for approximately 4 hours at 37°C under vigorous agitation. The digested adipose tissue samples were then filtered through a 70-μm nylon mesh cell strainer (BD Biosciences, Bedford, MA), and centrifuged at 500×g for 10 minutes at room temperature (RT). The pellets were re-suspended and cultured in the growth medium DMEM: F12 (Life Technologies) supplemented with 10% ## # # Figure 6 Growth curve analysis of TwiASCs and WtASCs. The cells were originally seeded at 500 cells/cm 2 in 56.7 cm 2 Nunc cell culture plates and cultured for 10 days. Fold increase in cell number was analyzed every 2 days throughout the culture period and was calculated by comparing the cell number at each time point to the original plating cell number. # means P< 0.05 vs WtASCs on day 6 and day 10 respectively, and ## means P< 0.001 vs WtASCs on day 8 (t-test, n=3).

Flow cytometry

The following antibodies were used to define the surface markers expressed by the ASCs: CD29-FITC (fluorescein isothiocyanate), CD34-FITC, CD106-PE (vascular cell adhesion molecule-1 (VCAM-1)), CD31-PE (phycoerythrin), CD45-PE, CD11-PE, and Sca1-PE (stem cell antigen-1). All of the antibodies were purchased from BD Biosciences. The ASCs were cultured to 70% confluence, trypsinized, pelleted, and re-suspended in 500 μl phosphate buffered saline (PBS) (Life Technologies). The cells were incubated with the antibodies for 30 minutes at RT, then washed with PBS, and analyzed by Cytomics FC500 (Beckman Coulter, Brea, CA). The results were analyzed with CXP analysis software (Beckman Coulter).

Colony forming unit assay

ASCs at passage 3 were seeded onto 56.7 cm 2 Nunc cell culture plates (Nalge Nunc International, Rochester, NY) in 5 replicates at a total of 100 cells per plate. Growth media was changed every 3-4 days. After 14 days, the cells were washed with PBS, stained with 3% crystal violet in 100% methanol for 30 minutes at RT, and then washed with deionized (DI) water at least 3 times to remove excess dye. All colonies greater than 2 mm in diameter were counted.

Differentiation

ASCs at passage 3 were seeded with a density of 6×10 4 cells/well on 6-well Nunc plates (Nalge Nunc International) and cultured to approximately 90% confluence before adipogenic and osteogenic differentiation media were added. Adipogenic differentiation medium was ASC growth medium supplemented with 5 μg/ml insulin, 50 μM indomethacin, 1 μM dexamethasone and 0.5 μM 3-isobutyl- The *levels of adipogenic and osteogenic differentiation were also quantitated. For the quantitation of adipogenic differentiation, the accumulated lipids were eluted with isopropanol after images were captured. The amount of Oil Red O was measured by recording the optical density (OD) of the solution at 584 nm. The results were normalized to the protein content of the samples with the BCA assay (Thermo Scientific, Rockford, IL). For the quantitative osteogenesis assay, the cells were de-stained, after images were taken, with 10% cetylpyridinium chloride (Sigma) for 30 minutes at RT. The amount of Alizarin Red was determined by measuring the OD of the solution at 584 nm. The results were normalized to the protein content of the samples.

Real-time PCR analysis of osteogenic markers in WtASC and TwiASC

Total RNA was extracted from differentiated WtASC and TwiASC on day 7, day 14, and day 21 using RNeasy Mini Kit (Qiagen, Valencia, CA). RNA concentration and purity were assessed by Nanodrop 2000C spectrophotometer (Thermo Scientific). First strand cDNA syntheses were performed using iScript cDNA Synthesis kit (Biorad, Hercules, CA) after RNA was first treated with DNase (Life Technologies). Primers for mouse alkaline phosphatase (ALP), Runx-2 (RUNX), and osteocalcin (OCN) were commercially synthesized (IDTDNA, Coralville, Iowa), and the sequence of the primers was as previously described [44]. The housekeeping gene β-actin (UniGene: Mm328431) was used as internal reference. The PCR reactions were performed using CFX96 Real Time System (Biorad) in a total final volume of 25 μl containing 12.5 μl SYBR green supermix (Biorad), 1 μl forward primer and 1 μl reverse primer (400 nM), and 1 μl template (500 ng). Reaction mixtures were incubated at 95°C for 3 minutes, and reactions were allowed to proceed via 45 cycles of melting at 95°C for 15 seconds, annealing and extension at 57°C for 30 seconds. Quantification was calculated using ΔΔCt method [44].

Growth curve

ASCs at passage 3 were seeded onto 56.7 cm 2 Nunc cell culture plates in triplicate with a seeding density of 500 cells/cm 2 . Growth medium was changed every 3-4 days. Every 2 days for a total of 10 days, the cells were trypsinized, pelleted, re-suspended in 500 μl PBS, and counted using a Countess W Automated Cell Counter (Life Technologies) to analyze the fold increase in cell number.

Macrophage and ASCs co-culture

Primary macrophages were obtained through thioglycolateelicited peritoneal isolation using a slightly modified published method [42]. Briefly, 1 ml 3% (w/v) sterile thioglycolate (Sigma) in DI water was injected intraperitoneally into C57Bl6 mice (Jackson Lab) to elicit peritoneal exudate cells. Four days after thioglycolate injection, cells were harvested by peritoneal lavage using 10 ml PBS, treated with red blood cell lysing buffer (Sigma) to remove the red blood cells, assessed for macrophage purity by flow cytometry using FITC-labeled IgG anti-CD11b (BD Biosciences), and finally plated onto 24-well Nunc cell culture plates (Nalge Nunc International) with 200,000 cells per well in RPMI medium (Life Technologies) supplemented with 10% heat inactivated FBS and 1% penicillin/streptomycin. After 6-hour incubation, the non-adherent cells were removed by vigorous washing with PBS. Then 0.4 μm pore size 24 well transwell inserts (Corning, Lowell, MA) were placed into the 24-well plate, and TwiASCs or WtASCs were plated in each transwell insert with 40,000 cells per well (ASC: macrophage ratio=1:5). Cells were cultured overnight, incubated another 24 hour after the addition of 30 ng/ml lipopolysaccharide (LPS, Sigma), and then total RNA was extracted from the macrophages using RNeasy Mini Kit. RNA concentration and purity were assessed by Nanodrop 2000C spectrophotometer.

Real-time quantitative PCR

RNA was first treated with DNase, and first strand cDNA syntheses were performed using iScript cDNA Synthesis kit. Real-time PCR assay was performed to analyze the levels of mouse inflammatory cytokines IL-1α, IL-1β, TNFα, IL-6 and IL-10 expressed by macrophages transwell co-cultured with TwiASCs and WtASCs. PCR analyses were performed using CFX96 Real Time System in a total volume of 20 μl containing 10 μl Taqman mastermix (Applied Biosystems, Foster City, CA), 1 μl primer and probe mix (Applied Biosystems), and 2 μl template. Reaction mixtures were incubated at 50°C for 2 minutes and 95°C for 10 minutes, and reactions were allowed to proceed via 40 cycles of melting at 95°C for 15 seconds, annealing and extension at 60°C for 1 minute. The housekeeping gene β-actin (UniGene: Mm328431) was used as internal reference. Quantification was calculated using ΔΔCt method [45]. Controls were RNA from macrophages cultured alone with and without LPS.

Statistical analysis

Groups of data were analyzed by one-way analysis of variance (ANOVA). For pair-wise comparisons, the F-test was used to determine whether a given pair of population variances was equal (α<0.05). This information was then used in designating the appropriate t-tests (typically heteroscedastic) to perform for comparing the means of population pair, with significance defined as P<0.05. All values were reported as mean± SD.

Figure 1
1Morphology and cell size of TwiASCs and WtASCs. A) Morphology of TwiASCs and WtASCs. Both cell types retain the thin spindle fibroblast-like morphology described for ASCs. B) Cell size based on forward scatter signal of flow cytometry (P=0.96, t-test, n≥10).

Figure 2
2Cell surface phenotype analysis of TwiASCs and WtASCs. A) Cells were analyzed by flow cytometry for MSC surface markers CD29, Sca1 and CD106; hematopoietic markers CD34 and CD45; phagocytic lineage marker CD11b; and endothelial marker CD31. Gray filled: isotype control; gray line: WtASCs; black line: TwiASCs. B) The percentage expression of each surface marker. All cells were analyzed at passage 3. # means P< 0.05 vs TwiASCs for CD34 (t-test, n=3).

Figure 3
3Qualitative and quantitative differentiation of TwiASCs and WtASCs along osteogenic and adipogenic lineages. A) Image of differentiated TwiASCs and WtASCs. Passage 3 TwiASCs and WtASCs were incubated in adipogenic or osteogenic differentiation media for 21 days and stained, respectively, with Oil Red O and Alizarin Red. For osteogenic differentiation, images were collected at 4× magnification. For adipogenic differentiation, images were obtained at 10× magnification. B) Quantification of osteogenesis and adipogenesis. For the quantitation of osteogenesis, the cells were de-stained with 10% cetylpyridinium chloride after stained with Alizarin Red. For the quantitation of adipogenesis, the cells were de-stained with isopropanol. Optical density (OD) was measured at 584 nm and normalized to protein content. The bar graph represents the ratio of normalized OD of differentiated cells and normalized OD of control cells. # means P< 0.05 vs WtASCs (t-test, n=3).

Figure 7
7The inflammatory cytokine profiles of LPS-stimulated macrophages. The ASCs and thioglycolate-elicited macrophages were transwell co-cultured and incubated for another 24 hours in the presence of 30 ng/ml LPS. The macrophage cells were collected, and total RNA was extracted for real-time PCR quantification of inflammatory cytokines IL-1α, IL-1β, TNFα, IL-6 and IL-10. The macrophage (MФ) alone and LPS stimulated macrophage (MФ+LPS) were used as controls. ## means P< 0.01 MФ+LPS vs MФ+LPS (WtASCs) (t-test, n=3); **means P< 0.01 MФ+LPS vs MФ+LPS (TwiASCs) (t-test, n=3); # means P< 0.05 MФ+LPS vs MФ+LPS (WtASCs) (t-test, n=3); and ¥ means P< 0.05 MФ+LPS(WtASCs) vs MФ+LPS (TwiASCs) (t-test, n=3).
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AcknowledgementsWe thank Alan Tucker in our flow cytometry core for his help with flow cytometry analysis. Funding for this study was provided by Tulane University and the Pennington Biomedical Research Foundation.Competing interestsThe authors declare that they have no competing interest.Authors' contributions XZ conceived of the study, performed all the experiments and drafted the manuscript. JS helped design the study and edited the manuscript. SZ helped the macrophage co-culture experiment. AL helped the differentiation quantification experiment and edited the manuscript. BS did the genotyping of Twitcher mice and edited the manuscript. JG edited the manuscript. BB supervised the entire study and edited the manuscript. All authors read and approved the manuscript.Submit your next manuscript to BioMed
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Cost of inappropriate antimicrobial use for upper respiratory infection in Japan

Shinya Tsuzuki
Yuki Kimura
Masahiro Ishikane
Yoshiki Kusama
Norio Ohmagari
Cost of inappropriate antimicrobial use for upper respiratory infection in Japan
10.1186/s12913-020-5021-1R E S E A R C H A R T I C L E Open Access
Background: Antibiotics are often prescribed inappropriately to patients with upper respiratory infection (URI) in ambulatory care settings; however, the economic burden of such prescription has not been quantitatively assessed. Here, we aimed to evaluate the additional cost of antimicrobial prescription for URI at the population level in Japan.Methods:We conducted a retrospective observational survey using longitudinal claims data between 2013 and 2016 obtained from JMDC Claims Database, which contains data from 5·1 million corporate employees and family members under the age of 65 years. Appropriateness of antibiotic prescription was assessed by a panel of six infectious disease physicians according to ICD-10 code in JMDC Claims Database. Total additional cost of antibiotic prescription for URI at the national level was estimated by weighting of age-structured population data.Results: The annual additional cost of inappropriate antibiotic prescription for URI was estimated at 423·6 (95% CI: 416·8-430·5) million USD in 2013, 340·9 (95% CI: 335·7-346·2) million USD in 2014, 349·9 (95% CI: 344·5-355·3) million USD in 2015, and 297·1 (95% CI: 292·4-301·9) million USD in 2016. Three classes of broad-spectrum oral antibiotics (third-generation cephalosporins, macrolides, and fluoroquinolones) accounted for > 90% of the total additional cost.Conclusions: Although a decreasing trend was observed, annual additional costs of inappropriate antibiotic prescriptions for URI could be a substantial economic burden in Japan. Appropriately prescribing broad-spectrum oral antibiotics might be an important issue to reduce unnecessary medical costs in Japanese ambulatory care.

Background

Antibiotic prescription promotes antimicrobial resistance (AMR), which is currently one of the greatest threats to global health [1]. Antibiotic consumption is one of the key drivers of AMR [2,3]; thus, reasonable prescription should be an important strategy of AMR countermeasures. Nevertheless, antibiotics are often prescribed inappropriately to patients with upper respiratory infection (URI) in ambulatory care settings [4,5].

Following the publication of the Global Action Plan on Antimicrobial Resistance in 2015 [1], the Japanese government established the National Action Plan on Antimicrobial Resistance in 2016 [6]. According to this plan, reduction of oral antibiotics is specified as one of the main outcome indices for the evaluation of AMR countermeasures in Japan. A previous study estimated that physicians have prescribed antibiotics for about 60% of URI cases [7]. However, the trend of prescription behaviour of inappropriate antibiotic use for URI in ambulatory care remains unclear.

Additionally, antibiotic prescription for URI might impose a significant burden on our society because although URI is not a severe disease at the individual level, the total cost of antimicrobial use for URI does not seem to be negligible. Furthermore, AMR caused by such antimicrobial use will result in additional AMR infections and these cases will impose significant additional costs.

Although there are some previous studies about the additional cost of antimicrobial prescription for URI in ambulatory care settings, these studies focused on more extensive kind of drugs or diseases or specific populations [8][9][10]. Therefore, the main objective of the present study is to clarify the amount of additional cost due to inappropriate antibiotic prescription for URI and its trend in recent years.

Methods

Data source

We conducted a retrospective observational survey using longitudinal claims data collected between 2013 and 2016 obtained from JMDC Claims Database, which contains anonymous, de-identified claims data of 5·1 million corporate employees who are covered by the employees' health insurance plan and their family members under the age of 65 years.

Assessment of appropriate antibiotic prescription

We identified the number of claims that included inappropriate antibiotic prescription for URI patients. URI patients were defined by claims with the International Classification of Diseases, 10th Revision (ICD-10) diagnosis code of J00-06 and/or J20-22 without other diagnosis considered as appropriate for antimicrobial treatment.

To judge appropriate antibiotic prescribing behaviour for other ICD-10 codes, a panel of six infectious disease doctors at the Japanese Disease Control and Prevention Center, National Center for Global Health and Medicine, assessed the appropriateness of antibiotic prescription for each ICD-10 code. Each physician first independently evaluated whether treatment with antibiotics was optimal. If two or more assessors did not reach an agreement for any diagnosis code, then all six experts reviewed the case at a round table until a consensus was reached. The validity of this approach has been shown in previous studies [11,12]. Details of the discussion process to judge appropriateness of antibiotic prescription are available in Supplementary file 1. As a result of the discussion, 75 ICD-10 codes are classified as "inappropriate" for antibiotic use. Details of ICD-10 codes classification are available in Supplementary file 2.

Cost analysis

The total additional cost of inappropriate antibiotic prescription at the national population level was estimated by the JMDC data, which include the duration and cost of each drug prescribed in each claim. The cost of inappropriate antibiotic prescription in each age group was collected, and each year was weighted according to Japanese age-stratified population structure based on 5year age group data [13]. We did not include cost of antibiotics assessed as appropriate. Costs of oral thirdgeneration cephalosporins, macrolides, and fluoroquinolones were separately evaluated. We did not consider the cost of treatment for side effects caused by unnecessary antibiotic prescription (e.g. anaphylaxis, urticaria, and diarrhoea). We assumed that each patient's diagnosis was appropriate and antibiotic prescription for URI did not treat any bacterial infection case that was misdiagnosed as URI.

To reflect uncertainty, we estimated 95% confidence intervals (CIs) of the proportion of population coverage, the proportion of URI cases prescribed antibiotics, and the cost of antibiotics in each case. We conducted the binomial test to estimate 95% CIs of population coverage and the proportion of antibiotic prescription. For the cost of antibiotics, we conducted the t-test to estimate 95% CIs.

All analyses were conducted by Stata MP15 [14] (for data aggregation) and R (for statistical analyses) version 3.5.2 [15]..

Role of the funding source

The funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. The first and corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication.

Results

Study population

The JMDC database covered about 3% of the total population of Japan under the age of 65 years (2·85% in 2013 and 3·45% in 2016). Details of enrollees are described in Table 1.

Proportion of antibiotic prescription for URI

The estimated proportion of inappropriate antibiotic prescription for URI in each year and age group is shown in Table 2. About 30% of URI cases in ambulatory care settings were prescribed antibiotics (32·41% in 2013 and 29·36% in 2016).

Cost of antibiotic prescription

The estimated additional cost of inappropriate antibiotic prescription for URI at the total Japanese population level is shown in Table 3. Total additional cost amounts were 300-400 million USD annually from the healthcare payer's perspective (423·6 million USD in 2013 and 297·1 million USD in 2016). Figure 1 describes the breakdown of the additional cost of antibiotic prescriptions by age group. About 30% of all antibiotic prescriptions for URI were for children under 15 years (30·2% in both 2013 and 2016).

Breakdown of antibiotic class

Annual estimated percentages and additional costs of antibiotic prescription for three broad-spectrum antibiotic classes (third-generation cephalosporins, macrolides, and fluoroquinolones) are shown in Fig. 2 and Tables 4 and 5. Third-generation cephalosporins were prescribed to about 12% of URI cases (13·17% in 2013 and 11·33% in 2016).

Macrolides were prescribed to about 10% of URI cases (11·06% in 2013 and 10·38% in 2016) and fluoroquinolones were prescribed to about 5% of URI cases (4·60% in 2013 and 4·23% in 2016). Additional prescription costs of these three antibiotic classes amounted to 403·9 million USD in 2013 and 280·0 million USD in 2016. Details of antibiotics prescribed for URI by class are available in Supplementary file 3.

We also evaluated the share of broad-spectrum antibiotics in the total annual cost of antibiotic prescription. Three antibiotic classes (third-generation cephalosporins, macrolides, and fluoroquinolones) accounted for over 90% of total additional antibiotic prescription costs for URI (95·3% in 2013 and 94·2% in 2016).

Discussion

We developed a national population level cost estimation about the unnecessary cost of antibiotic prescription
0-4 22·45 (22·37-22·52) 21·59 (21·52-21·66) 20·14 (20·07-20·21) 18·87 (18·80-18·94) 5-9 30·92 (30·79-31·05) 30·37 (30·25-30·50) 28·32 (28·20-28·44) 26·75 (26·63-26·87) 10-14 36·88 (36·69-37·08) 35·95 (35·76-36·13) 34·56 (34·39-34·74) 33·04 (32·86-33·22) 15-19 43·29 (43·0-43·58) 42·02 (41·75-42·30) 41·71 (41·45-41·97) 37·87 (37·63-38·12) 20-24 44·05 (43·74-44·36) 42·72 (42·42-43·01) 43·61 (43·32-43·91) 40·49 (40·21-40·78) 25-29 42·51 (42·24-42·78) 41·68 (41·42-41·94) 42·09 (41·84-42·35) 39·99 (39·73-40·24) 30-34 42·74 (42·51-42·96) 42·01 (41·79-42·23) 41·52 (41·30-41·74) 39·80 (39·58-40·02) 35-39 42·82 (42·61-43·04) 42·09 (41·88-42·30) 41·45 (41·24-41·66) 39·98 (39·77-40·19) 40-44 41·6 (41·37-41·82) 40·95 (40·74-41·17) 40·46 (40·26-40·67) 39·04 (38·83-39·25) 45-49 39·10 (38·85-39·35) 38·16 (37·93-38·40) 38·40 (38·17-38·62) 36·87 (36·65-37·10) 50-54 36·75 (36·49-37·01) 36·31 (36·06-36·56) 36·09 (35·85-36·33) 34·79 (34·55-35·02) 55-59 35·45 (35·16-35·74) 34·88 (34·60-35·15) 35·04 (34·78-35·30) 33·45 (33·20-33·71) 60-64 33·62 (33·30-33·94) 33·06 (32·74-33·38) 33·27 (32·96-33·58) 31·71 (31·40-32·02) Total 32·41 (32·37-32·47) 31·68 (31·63-31·73) 31·0 (30·95-31·05) 29·36 (29·31-29·40) a
Data are expressed as percentages. Values in parentheses represent 95% confidence intervals for URI. To our knowledge, the present study is the first to assess physicians' antibiotic prescription behaviour not only in view of the proportion of antibiotic prescriptions but also the associated additional costs. By adding the viewpoint of cost, our findings show novel characteristics of inappropriate antibiotic prescription in ambulatory care settings. First, both the proportion and cost of unnecessary antibiotic prescription for URI have decreased in recent years. The proportion of URI cases to which physicians prescribed antibiotics was 32·41% in 2013 but only 29·36% in 2016. The cost of antibiotic prescriptions for URI cases was 423·6 million USD in 2013 and decreased to 297·1 million USD in 2016.

The proportion of URI cases to which physicians prescribed unnecessary antibiotics demonstrated a gradually decreasing trend every year and the total annual cost of antibiotic prescriptions for URI in 2016 was about 30% lower than that in 2013. According to a previous study, Japanese physicians had prescribed antibiotics for 60% of URI cases in 2005 [7]. It is difficult to compare the results of the present study and that of the previous study because of sample size (the previous study analysed only 2577 claims), sampling period (the previous study collected claims from January to March 2005 only), and other factors. Nevertheless, this decreasing trend of unnecessary antibiotic use might have already existed in Japan early in this century.
0-4 4 0 ·3 (39·9-40·7) 32·7 (32·4-33·0) 27·2 (26·9-27·5) 22·1 (21·8-22·3) 5-9 5 6 ·6 (55·9-57·3) 50·1 (49·5-50·7) 51·1 (50·5-51·8) 43·4 (42·8-43·9) 10-14 31·2 (30·7-31·7) 27·7 (27·3-28·1) 29·0 (28·6-29·4) 24·4 (24·0-24·7) 15-19 19·5 (19·1-19·8) 14·6 (14·4-14·8) 16·1 (15·8-16·4) 14·0 (13·8-14·3) 20-24 17·2 (16·9-17·5) 11·4 (11·2-11·6) 11·1 (11·0-11·3) 9·5 (9·3-9·6) 25-29 22·4 (22·0-22·7) 16·1 (15·8-16·4) 15·5 (15·3-15·8) 12·6 (12·4-12·8) 30-34 31·2 (30·8-31·7) 23·9 (23·6-24·2) 23·9 (23·5-24·2) 19·8 (19·5-20·1) 35-39 38·0 (37·4-38·5) 29·9 (29·5-30·3) 29·6 (29·2-30·0) 23·7 (23·4-24·1) 40-44 39·7 (39·1-40·2) 34·9 (34·4-35·4) 37·6 (37·1-38·1) 30·5 (30·1-30·9) 45-49 31·9 (31·3-32·4) 28·4 (28·0-28·9) 31·8 (31·3-32·3) 30·3 (29·8-30·8) 50-54 29·4 (28·8-29·9) 26·2 (25·7-26·7) 30·0 (29·5-30·6) 26·2 (25·7-26·7) 55-59 31·1 (30·4-31·8) 23·0 (22·5-23·4) 25·3 (24·8-25·8) 23·0 (22·6-23·5) 60-64 35·4 (34·5-36·3) 22·0 (21·5-22·6) 21·7 (21·2-22·2) 17·7 (17·3-18·2) Total 423·6 (416·8-430·5) 340·9 (335·7-346·2) 349·9 (344·5-355·3) 297·1 (292·4-301·9) a
Values in parentheses represent 95% confidence intervals As mentioned above, the Japanese government established the National Action Plan on AMR in 2016 [6]. While it will take a few more years to assess the impact of this plan because it is newly established, our findings might reflect physicians' alteration in awareness on AMR rather than behavioural change in antibiotic prescription.

Conversely, the proportion of antibiotic prescriptions for URI is difficult to compare with previous studies from foreign countries due to differences in settings and conditions. For example, Barnett and Linder reported that the antibiotic prescribing rate dropped from roughly 80 to 70% around 1993 and decreased again around 2000 to 60% [16]. However, their study was limited to adults and 'sore throat' cases; therefore, a large number of bacterial pharyngitis cases were likely included. Finkelstein and colleagues examined the effect of an educational outreach intervention in ambulatory care but only evaluated paediatric cases [17]. Fleming-Dutra and colleagues conducted a more extensive analysis, however, their focus was on whole ambulatory care, which includes diagnoses sometimes appropriate for antibiotic prescription (e.g. otitis media and sinusitis).

Furthermore, we must take the differences in healthcare system between Japan and other countries into consideration. The Japanese healthcare system generally secures 'free access' to healthcare services, regardless of facility level, and there is no limitation on the frequency of ambulatory care visit [18]. This implies that a larger number of URI patients tends to visit ambulatory care although URI is basically a self-limited disease and there are substantial numbers of URI patients amongst patients admitted to healthcare facilities [19,20]. This fact makes it more difficult to compare the proportion of URI cases prescribed antibiotics prescribed. While decreasing trends in the proportion and cost of antibiotic prescription for URI are favourable findings, the main driver of this trend and the current situation of Japanese ambulatory care in global society are still not clear.

Second, our results demonstrated the importance of appropriate antibiotic use, especially in paediatric ambulatory care. Despite the comparatively low rate of antibiotic prescription in children and the ratio of children to the total population, the additional cost in children under 15 years accounted for over 30% of total additional costs annually. This finding likely reflects the frequency of healthcare facility visits attributed to URI by children [21]. Additionally, most Japanese nursery schools and kindergartens require caregivers to bring their children to healthcare facilities when they catch a Values in parentheses represent 95% confidence intervals cold [22]. As a result, children are exposed to the risk of unnecessary antibiotic prescription more frequently than adults are in Japanese ambulatory care. Therefore, children should be an appropriate target population for interventions to promote appropriate antibiotic use. Third, it is noteworthy that broad-spectrum oral antibiotics accounted for the majority of additional antibiotic prescription costs for URI. Three antibiotic classes (third-generation cephalosporins, macrolides, and fluoroquinolones) comprised almost 95% of total additional antibiotic prescription costs for URI in Japan. Our finding of a high prescription rate of third-generation cephalosporins and macrolides is compatible with findings from a previous study [23]. The fluoroquinolone prescription rate was comparatively low, however, it can be attributed to the clinical contraindication of fluoroquinolones for children. We observed an extremely low prescription rate of fluoroquinolones in children under 5 years and a high rate in adults (Supplementary file 3). Considering these findings with the difference in drug prices between these broad-spectrum antibiotics and other narrow-spectrum antibiotics (e.g. penicillins), we can understand the significance of appropriate use of broad-spectrum antibiotics more profoundly. Compared with other high-income countries, Japan demonstrated higher consumption rates of these three antibiotic classes [24]. Namely, these broad-spectrum antibiotics could be an appropriate target for antimicrobial stewardship in ambulatory care in Japan, not only from the standpoint of antimicrobial resistance but also that of economic burden.

Strengths and limitations

A major strength of the present study is the use of a large number of individual patient-level claims data, which cover both children and adults. Conversely, our data did not include claims of patients 65 years of age or older.

While millions of individual claims are included in the dataset, its representativeness is not completely assured because our data are based on information from health insurance purchased by enterprises. Nevertheless, a low proportion of self-employed people (8·48% in 2017) and low unemployment rate (3·1% in 2016) in Japan [25] enable us to justify the result of our analyses. Because selfemployed and unemployed people are also covered by other types of national health insurance in the Japanese healthcare system and out-of-pocket costs are reimbursed according to each individual's income, we can expect that healthcare-seeking behaviour would not be greatly different among employed, self-employed, and unemployed individuals. Nonetheless, further study would be desirable to examine the difference in healthcare-seeking behaviour brought by employment status.

In addition, diagnoses in the dataset are sometimes unreliable. It is often the case that physicians make different diagnosis in the claims on purpose to justify their examinations and prescriptions. Nevertheless, physicians never make a fake diagnosis of "URI" when they would like to prescribe antibiotics, but make a diagnosis of other bacterial infections in order to justify their antibiotic prescriptions. Then therefore unreliable diagnoses might not overemphasize the cost of inappropriate antibiotic prescription.

Another strength is that we introduced the concept of cost. Although the rate of antibiotic prescription or defined daily dose [26] are indicators understood intuitively, our findings added another aspect of broadspectrum antibiotics. As one important limitation, our present analyses showed conservative results because we did not consider any additional cost of adverse effects of antibiotics. For example, rush, diarrhoea, and anaphylaxis are general adverse effects sometimes observed with systemic use of oral antibiotics. Of course, these adverse events might impose additional medical costs, however, there is no appropriate information about costs and the probability of such events in Japan thus far. Furthermore, broad-spectrum antibiotics have their own adverse effects. For example, oral third-generation cephalosporins can cause hypocarnitinaemia on rare occasions [27]. Azithromycin is associated with a slightly increased risk of cardiovascular death [28]. Fluoroquinolones uncommonly cause tendon rupture [29] and QT prolongation
101·7 (99·3-104·1) 108·1 (105·6-110·5) 93·4 (91·2-95·6) Fluoroquinolones 135·4 (131·2-139·7) 108·1 (104·9-111·4) 111·2 (107·9-114·6) 93·5 (90·6-96·5) Total 403·9 (394·8-414·3) 323·9 (316·1-331·8) 333·1 (325·0-341·2) 280·0 (273·5-287·8)
a Values in parentheses represent 95% confidence intervals [30] and the US Food and Drug Administration updated its drug safety information for fluoroquinolone due to the rare but serious risk of aortic ruptures or tears in certain patient [31]. If we can take these adverse events into consideration as a form of medical cost, then the estimated economic burden of antibiotic prescription for URI might be more precise.

Conclusions

As we have shown, the present study gives an extensive understanding about the impact of inappropriate antibiotic prescription for URI. Although a decreasing trend was observed, the annual additional cost of antibiotic prescription for URI could be considered a substantial economic burden in Japan. Our study also suggests that appropriate use of broad-spectrum oral antibiotics might be critical to reduce unnecessary medical costs in Japanese ambulatory care.

Supplementary information

Supplementary information accompanies this paper at https://doi.org/10. 1186/s12913-020-5021-1.

Additional file 1. Supplementary Spreadsheet. Details of the discussion process to judge appropriateness of antibiotic prescription. Table. Appropriateness of antibiotic prescribing for ARTI used in the study.

Additional file 2. Supplementary

Additional file 3. Table S1. Estimated percentage of third-generation cephalosporin prescriptions for URI in ambulatory care. Table S2. Estimated additional cost of third-generation cephalosporin use for URI in ambulatory care. Table S3. Estimated percentage of macrolide prescriptions for URI in ambulatory care. Table S4. Estimated additional cost of macrolide use for URI in ambulatory care. Table S5. Estimated percentage of fluoroquinolone prescriptions for URI in ambulatory care.

Fig. 1
1Additional cost of antibiotic prescription for URI in children and adultsTsuzuki et al.

Fig. 2
2Breakdown of annual additional cost of antibiotic prescription by antibiotic class. 3GC, third-generation cephalosporins; FQ, fluoroquinolones; MC, macrolidesTable 4 Estimated percentages of broad-spectrum antibiotic prescription in ambulatory care (4·58-4·62) 4·45 (4·43-4·47) 4·55 (4·53-4·57) 4·23 (4·21-4·26) a

Table 1
1Number of enrollees in each age group aValues in parentheses represent the proportion of enrollees of total number of Japanese populations in each age groupAge group (years)
2013
2014
2015
2016
Total
2,719,847 (2·85)
3,266,315 (3·47)
3,210,652 (3·44)
3,185,191 (3·45)

a

Table 2
2Estimated percentage of inappropriate antibiotic prescription for URI in ambulatory care aAge group (years)
2013
2014
2015
2016

Table 3
3Estimated additional cost of antibiotic use for URI in ambulatory care (unit = million USD) aAge group (years)
2013
2014
2015
2016

Table 5
5Estimated additional costs of broad-spectrum antibiotic prescription in ambulatory care (unit = million USD) aClass
2013
2014
2015
2016

Third-generation cephalosporins
134·7
(132·0-137·5)

114·1
(111·9-116·3)

113·8
(111·5-116·1)

93·7
(91·7-95·7)

Macrolides
133·8
(130·6-137·1)

Table S6 .
S6Estimated additional cost of fluoroquinolone use for URI in ambulatory care. 3GC: Third-generation cephalosporins; AMR: Antimicrobial resistance; CI: Confidence interval; FQ: Fluoroquinolones; ICD-10: International Classification of Diseases, 10th Revision; JPY: Japanese yen; MC: Macrolides; URI: Upper respiratory infection; USD: United States dollarAbbreviations

AcknowledgementsWe would like to thank JMDC Inc. for data provision. We also thank Christina Croney, PhD, from Edanz Group (www.edanzediting.com/ac), for editing a draft of this manuscript.Authors' contributions ST conceived and designed the study. YK1, YK2, MI, and NO acquired the data. YK1 and ST analysed the data and all authors interpreted the results. ST drafted the first version of the manuscript. YK1, YK2, MI, and NO critically revised the manuscript. All authors approved the final version of the manuscript. The corresponding author attests that all listed authors meet authorship criteria and that no others meeting the criteria have been omitted.FundingThis study was supported by a Ministry of Health, Labour and Welfare (MHLW) research grant of Japan (H29-shinkougyousei-shitei-005). Funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Availability of data and materialsThe datasets for the study are not publicly available due to company policy of JMDC Inc. Data are, however, available from the corresponding author upon reasonable request and with the permission of the JMDC Inc.Ethics approval and consent to participate Not applicable because no individual data in any form have been reported in this article.Consent for publicationNot applicable.Competing interestsAll authors declare no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous 3 years; and no other relationships or activities that could appear to have influenced the submitted work.Author details 1
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