Enhanced Production of Early Lineages of Monocytic and Granulocytic Cells in Mice with Colitis

Enhanced production of early lineages of monocytic and granulocytic cells in mice with colitis

Mark D. Trottiera,1, Regina Irwinb, Yihang Lia,2, Laura R. McCabeb, and Pamela J. Frakera,3

Departments of aBiochemistry and Molecular Biology and bPhysiology, Michigan State University, East Lansing, MI 48824

Contributed by Pamela J. Fraker, August 28, 2012 (sent for review April 26, 2012) The bone marrow (BM) is a large, highly active, and responsive numbers in the stools, being used in the past as one method for tissue. Interestingly, little is known about the impact of colitis on determining the pathology of the various forms of IBD (12). hematopoietic functions. Using dextran sodium sulfate (DSS) to Therefore, understanding how forms of IBD affect the production induce colitis in mice, we identified significant changes in the BM. of these cells in the marrow was also important to understanding Specifically, cells of the monocytic and granulocytic lineages in- the etiology of this disease. creased nearly 60% and 80%, respectively. This change would sup- Although CD and UC are categorized as autoimmune diseases, port and promote the large infiltration of the gut with neutrophils it is known that modifying the epithelial barrier, for example and monocytes that are the primary cause of inflammation and tis- through modulation of β-catenin, causes a Crohn’s-like colitis (13). sue damage during colitis. Conversely, the early lineages of B and T Studies in senescence accelerated mouse P-1 (SAMP) mice, which cells declined in the marrow and thymus with particularly large los- display ileitis similar to Crohn’s disease, also suggest that defects in ses observed among pre-B and pre-T cells with heightened levels of epithelial permeability may be the primary source of ileitis sus- + + apoptosis noted among CD4 CD8 thymocytes from DSS-treated ceptibility in these mice and that inflammatory responses occur mice. Also noteworthy was the 40% decline in cells of the erythro- as a consequence of this initial barrier break (14). Therefore, to cytic lineages in the marrow of colitis mice, which undoubtedly con- carefully assess how changes in barrier function impact hemato- tributed to the anemia observed in these mice. The peripheral blood poiesis, we used a DSS mouse model of colitis. DSS treatment reflected the marrow changes as demonstrated by a 2.6-fold increase induces colitis by increasing gut epithelial barrier permeabil- in neutrophils, a 60% increase in monocytes, and a decline in the ity, which is thought to allow gut bacteria to activate immune lymphocyte population. Thus, colitis changed the BM in profound responses and promote colitis. The data will show that colitis IMMUNOLOGY ways that parallel the general outcomes of colitis including infiltra- made substantial changes in lymphopoiesis in both the marrow tion of the gut with monocytes and neutrophils, inflammation, and and the thymus with major losses in pre-T and pre-B cells within anemia. The data provide important understandings of the full im- 15 d. Likewise, there were substantial reductions in erythrocytic pact of colitis that may lead to unique treatments and therapies. lineages that would contribute to and extend the anemia in DSS- treated mice. Conversely, cells of the monocytic and granulocytic granulopoiesis | lymphopoiesis | inflammatory bowel disease lineages increased significantly in the marrow and peripheral blood of mice with colitis, which could promote or sustain in- lcerative colitis (UC) and Crohn’s disease (CD) are chronic flammation and tissue damage in the gut. These important find- Uinflammatory bowel diseases (IBD) that affect more than 1.4 ings clearly show that colitis significantly alters hematopoietic million Americans (1). Both genetic and environmental factors processes. contribute to the hyperactivity and dysregulation of the immune system associated with colitis. IBD is characterized by recurrent Results bouts of illness associated with symptoms of weight loss, diarrhea, Confirmation of Disease in Mice. Mice, 6 wk of age, were separated anemia, rectal bleeding, and abdominal pain (1, 2). Past studies into two weight-matched groups (22.4 ± 1.4 g for controls and demonstrate that gut infiltration with neutrophils, monocytes, and 22.6 ± 1.6 g for colitis mice). The colitis group received 1% DSS macrophages is a major contributor to the gastrointestinal tissue in drinking water, whereas controls received regular drinking injury and inflammation noted in IBD (3–7). water. After 15 d, DSS-treated mice lost 1.8 ± 1.8 g (4.7%) of Although many facets of immune function in the IBD patient body weight, whereas controls gained 1.1 ± 0.9 g (8.0%), and in animal models have been investigated, almost nothing was resulting in an 11.1% lower body weight for DSS-treated mice known of the impact of IBD on the bone marrow (BM). In par- compared with controls (Fig. S1A). By 8 d, the majority of DSS- ticular, it was not known whether the ability of marrow to produce treated mice showed signs of rectal bleeding and soft fecal pel- fi new leukocytes or red blood cells each day was altered by UC or lets. DSS-treated mice displayed signi cant histologic changes in B CD. This question was of interest because the marrow is a large, cecums (Fig. S1 ) including structural changes marked by longer ± active tissue that produces billions of new cells each day and is and distorted crypt structure (scored at 4 1.3 compared with ± often impacted by changes in nutriture, stress, and disease (8–10). 1.2 1.0 for control mice), hyperchromasia, and few goblet cells Indeed, it was known that UC changed the rate of maturation of marrow osteoblasts, which contributed to the thinning of bones (11). Thus, the purpose of the research reported herein was to Author contributions: M.D.T., R.I., L.R.M., and P.J.F. designed research; M.D.T., R.I., Y.L., L.R.M., and P.J.F. performed research; M.D.T., R.I., and L.R.M. contributed new reagents/ determine the impact of dextran sodium sulfate (DSS)-induced analytic tools; M.D.T., R.I., Y.L., L.R.M., and P.J.F. analyzed data; and M.D.T., R.I., Y.L., L.R.M., colitis on hematopoietic function, with a focus on erythropoiesis, and P.J.F. wrote the paper. lymphopoiesis, and myelopoiesis (the production of neutrophils The authors declare no conflict of interest. and monocytes). Freely available online through the PNAS open access option. Myelopoietic processes in the marrow were of particular interest 1 fi Present address: College of Human Medicine, Michigan State University, Grand Rapids, because neutrophils are among the rst cells to be found in mucosa MI 49503. – of the gut, especially in the case of UC (3 5, 7). They secrete large 2Present address: Geriatric Research, Education and Clinical Center, VA Palo Alto Health amounts of chemokines that subsequently cause increasing num- Care System, Palo Alto, CA 94304. bers of monocytes/macrophages to enter these areas, creating 3To whom correspondence should be addressed. E-mail: [email protected]. fl – sustained injury and in ammation (3 5, 7). Indeed, cells of the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. myeloid lineages become so prevalent that they are found in large 1073/pnas.1213854109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1213854109 PNAS Early Edition | 1of6 Downloaded by guest on September 26, 2021 with colitis or enhanced elimination of lymphocytes from marrow via cell death and/or egress to peripheral sites (see Fig. S2 for typical flow cytometry plots; refs. 8 and 15). The erythrocyte compartment was reduced from 16.3% to 9.8% (down 40%) of nucleated cells. These data are particularly noteworthy given that anemia often accompanies colitis, and DSS treatment of rodents is known to cause anemia (16–19). Indeed, in blood of DSS-treated mice, significantly fewer red blood cells, reduced hemoglobin, and a lower hematocrit was observed, indicative of the beginnings of anemia (Table S1). Of note, colitis did not change the total number of nucleated cells within the marrow [38.0 ± 6.1 vs. 37.0 ± 5.0 (×106) in control versus colitis mice, respectively]. Thus, the deficits in cells of the lymphoid and erythroid lineages represented absolute declines in cell numbers, not just changes in the proportion of cells present. Conversely, increases in the number and proportion of cells of the myeloid lineages (monocytes, granulocytes) were noted in colitis mice. Monocytes increased more than 58%, from 9.8% of nucleated marrow cells in controls to 15.5% of cells in DSS mice. Likewise, granulocytes in marrow increased from 34% to 60% in DSS mice, an almost 80% increase (Fig. 1). These increases in cells of the myeloid lineages represent significant changes in the composition of the marrow and, therefore, its activity. These increases are also an important facet in the etiology of colitis that has been overlooked. It should be noted that the mixed progenitor population also declined nearly 40% in DSS-treated mice (Fig. 1). Because the composition of this group was not determined here, its potential impact if any is unknown. Because of the large decline in the lymphocyte compartment, the BM B-cell subpopulations were further examined. This dis- covery is important because in adults the majority of developing lymphocytes in the marrow are of the B lineage. We found that the B-cell population as a whole declined 70% in mice with colitis, representing a substantial decline in lymphopoiesis. Much of the decline in the marrow B-cell population was due to the loss of the pre–B-cell subset, which showed decreases in both Fig. 1. Changes in BM cellular distribution in control mice (Upper) and DSS- large and small pre-B cells as a proportion of the residual B-cell treated mice (Lower). Data were obtained by phenotypic labeling of BM cells population (71% and 24% decreases, respectively; Table 1). By with anti–Ly-6C and anti-CD31. Lymphoid cells, Ly-6C−/CD31+; erythroid cells, contrast, late pro-B cells more than doubled in proportion. A − − + − Ly-6C /CD31 ; monocytes, CD31 /Ly-6Chi; granulocytes, CD31 /lo/Ly-6Cmed; 44% increase in mature B cells was also observed, whereas the + mixed progenitors, CD31 /Ly-6Cmed (Fig. S2). Results shown are mean per- proportion of immature B cells modestly declined (Table 1). cent of total BM nucleated cells ± SD. One representative experiment of Thus, pro-B cells and immature-mature B cells were surviving = = < three is shown. Controls, n 10; DSS-treated, n 11 (*P 0.001). somewhat better than pre-B cells, which is not surprising because these cells are often more resistant to stress because of expres- (Fig. S1C). Consistent with disease, inflammatory cells were el- sion of anti-apoptotic Bcl-2 proteins. These proteins are lacking evated in the cecum lamina propria of DSS-treated mice, with in pre-B cells undergoing Ig gene rearrangement (20), suggesting – higher numbers of lymphoid aggregates (1.4 ± 0.7 compared with that apoptosis, which is ongoing in the pre B-cell lineages, might 0.4 ± 0.7 for control mice), a greater lymphoid cell score (5.2 ± be accelerated in DSS-treated mice. This change was the case 1.2 versus 1.4 ± 1.6 in controls), and the presence of neutrophils. The number of mucosal ulcers, a sign of disease severity, in- Table 1. Phenotypic distribution of bone marrow B cells creased threefold in colitis compared with controls. The ulcers † were moderate but in some cases extended into the submucosa. Sample Control ,% DSS-treated mice, % The overall disease score (the combination of parameters noted ‡ ± ± fi Pre Pro-B cells 3.9 0.3 2.6 0.9** above) was signi cantly higher for colitis compared with control Late Pro-B cells 10.7 ± 1.2 23.6 ± 3.0*** D fi mice (Fig. S1 ). The signi cant DSS-induced elevation of levels Large Pre-B cells 11.0 ± 1.3 3.2 ± 1.6*** β α of IL-1 , TNF- , and IL-6 mRNA in the proximal colon further Small Pre-B cells 50.0 ± 2.2 37.9 ± 6.0** fl E demonstrates in ammation/colitis (Fig. S1 ). Immature B cells 12.0 ± 1.0 10.2 ± 0.9** Mature B cells 4.3 ± 0.5 6.2 ± 3.3 Impact of Colitis on Hematopoietic Processes in the Marrow. Colitis had a significant effect on the composition of hematopoietic cells in **P < 0.01 the BM, causing changes in the proportion of cell lineages (Fig. 1). ***P < 0.001. † + ± The largest affect was seen in lymphocytic cells, which were sub- Data shown is percent of B220 population (26.2 2.3% of nucleated marrow for controls, 8.0 ± 1.6% for DSS-treated mice). ‡ + − − − + − stantially reduced by colitis. DSS treatment caused a 70% decline Pre Pro-B cells (B220 Gr-1 CD43hi IgM CD19 ); Late Pro-B cells (B220 Gr-1 − + + − − − in marrow lymphocytes, from nearly 26% of the total marrow cells CD43hi IgM CD19 ); Large Pre-B cells (B220 Gr-1 CD43 /low IgM FSChigh); + − − − + in controls to only 7.7% in DSS mice. This result represents either Small Pre-B cells (B220 Gr-1 CD43 /low IgM FSClow); Immature B cells (B220 a decline in the production and maturation of lymphocytes in mice Gr-1− CD43−/low IgM+ IgD−); Mature B cells (B220+ Gr-1− CD43−/low IgM+ IgD+).

2of6 | www.pnas.org/cgi/doi/10.1073/pnas.1213854109 Trottier et al. Downloaded by guest on September 26, 2021 Table 2. Numbers of CD4/CD8 subsets in thymus of control and DSS-treated mice − − + + + − − + Sample Total Thy (×106) CD4 /CD8 (×106) CD4 /CD8 (×106) CD4 /CD8 (×106) CD4 /CD8 (×106)

Control† 76.4 ± 23.1 5.16 ± 1.2 63.6 ± 19.9 2.55 ± 0.73 4.96 ± 1.5 ‡ DSS 33.1 ± 21.9 2.85 ± 1.5 26.5 ± 18.5 1.27 ± 0.67 2.39 ± 1.5 Change, % −56.7** −44.8 −58.3** −50.1** −51.8**

**Thy, thymocytes; P < 0.01. † Control (n = 10) and mice fed 1% DSS in drinking water for 2 wk (n = 11) were used. ‡Data shown is number of thymocytes in each population per mouse ± SD (× 106).

for pre-T cells developing in the thymus of DSS-treated mice, as the proportion of cells undergoing apoptosis being 38% higher will be seen. than the rate of death observed in control thymocytes. This sort of increase over time could certainly create the greater losses noted Impact of DSS on Thymopoietic Processes. To complete the de- in this population. There was no significant change in the apo- − − + piction of the impact of DSS on the primary immune tissues ptosis rate of CD4 CD8 precursors or more mature CD4 or + involved in lymphopoiesis, the thymus was investigated to com- CD8 T cells in the thymus of either group (Fig. 2). Thymocyte pare the observed decline in B-cell genesis in the BM. The av- apoptosis can be induced by elevated glucocorticoid levels; how- erage control thymus weighed 48 mg, whereas that of colitis mice ever, analysis of serum corticosterone in DSS-treated mice showed had atrophied to 28 mg. The total number of thymic cells lost no significant changes compared with controls at this point in time was ∼43 million in DSS-treated mice, dropping from 76.4 ± 23 (Fig. S3). In sum, apoptosis contributed to the observed thymic + + million present in controls to 33.1 ± 22 million in colitis mice atrophy and losses of CD4 /CD8 cells during colitis. (Table 2). This change represented a 57% decline in the number of cells in the thymus. It was of interest to investigate the dis- Composition of Peripheral Blood. To ascertain whether the changes tribution of T-cell subpopulations within the remaining thymo- observed in the BM and thymus were reflected in the periphery, + + cyte population. Phenotypic analysis showed the CD4 /CD8 differential counts of blood from control and DSS-treated mice thymocytes in DSS mice had declined approximately 60% in were evaluated (Table 4). In control mice, lymphocytes repre-

absolute numbers of cells (Table 2). Similar to the case of pre-B sented 80% of the leukocyte population, declining in DSS mice to IMMUNOLOGY cells, DSS treatment also caused substantial losses in the pro- 56% (Table 4). This result suggests that the decline in the lym- + + portion of pre-T (CD4 /CD8 ) cells, which declined to 75% of phopoietic compartments of the marrow and thymus had trans- the residual pool of cells (Table 3). However, similar to more lated into declining numbers of circulating lymphocytes. This mature B-cell populations, modest increases were noted in the outcome of course could compromise immune defense against − + + − proportion of mature CD4 /CD8 and CD4 /CD8 T cells, and infections. Likewise, the substantial increase in myeloid cells in − − a 50% increase was noted in early CD4 /CD8 T-cell precursors DSS-treated mice was reflected by a 2.6-fold increase in the per- (Table 3), although overall their absolute numbers had declined centage of circulating neutrophils and a 60% increase in blood (Table 2). This result is therefore analogous to the findings for monocytes (Table 4). This finding was surprising given the study changes in distribution of early B cells, because the better sur- was only of 15 d of duration. The total white blood cell count in viving cells have antiapoptotic proteins. DSS-treated mice did not significantly change. Thus, the enhanced − − Subdividing the latter group (CD4 /CD8 thymocytes) into proportion of cells of the myeloid lineages noted in the marrow double-negative (DN) populations, e.g., DN1–4, did not reveal was reflected by real increases in peripheral blood. Increased any noteworthy shifts in the proportion of these subpopulations blood neutrophils and monocytes indicates that cells were mi- (Table S2). Overall, DSS treatment caused an almost 60% de- grating out of the marrow in large numbers, thereby adding to cline in the number of cells in the thymus, with the greatest loss infiltration of the gut of mice with colitis, exacerbating and + + in absolute number of cells noted among the CD4 /CD8 cells. extending the ongoing inflammation. As discussed, the gut of DSS-treated mice was heavily infiltrated with neutrophils, whereas Underlying Cause of Reduced Thymic Lymphopoiesis. The large loss none were readily visible in control mice. in early lineages of lymphocytes in the primary immune tissues suggested the changes in DSS-treated mice might have accelerated Discussion apoptosis among these vulnerable precursor cells. To assess this Marrow is found throughout the skeletal mass of the body in- possibility, thymocytes from control and DSS mice were cultured cluding arms, legs, ribs, sternum, and skull. Collectively, the to determine apoptosis rate. Because the thymus contains large marrow represents a huge mass of tissue that is almost always + + percentages of CD4 CD8 pre-T cells (∼80%), known to be affected by disease but often ignored in the scientific literature. vulnerable to apoptosis, these cells were selected for analysis of The marrow no doubt requires a sizeable amount of nutrients potential changes in degrees of apoptosis. Fig. 2 shows more ap- to replenish cells that are needed daily by the billions. It was + + optotic cells among CD4 CD8 cells of DSS-treated mice, with already known that malnutrition, especially deficits in zinc or protein calories, substantially altered hematopoietic processes in the marrow, while initiating rapid thymic atrophy (9, 21). Table 3. CD4/CD8 subsets as a proportion of total thymocytes Moreover, stress levels of endogenously produced glucocorti- − − + + + − − + Sample CD4 /CD8 CD4 /CD8 CD4 /CD8 CD4 /CD8 coids also alter marrow function and cause thymic atrophy, in part due to initiation of apoptosis among pre-T and pre-B cells † ‡ Control , % 7.06 ± 1.2 83.0 ± 1.4 3.35 ± 0.32 6.48 ± 0.68 (8, 22). If one considers the known outcomes of colitis, which DSS§, % 10.7 ± 4.4 75.4% ± 11.5 5.17 ± 3.2 8.73 ± 4.1 includes weight loss, anemia, stress, reduced assimilation of Change, % 51.6** −9.16 54.3 34.7 nutrients, and accelerated numbers of myeloid cells in the gut **P < 0.05. and stools, it seemed highly probable that colitis would alter † Control (n = 10) and mice fed 1% DSS in drinking water for 2 wk (n = 11) BM functions. were used. The severity of colitis can vary depending on a variety of factors ‡ Data shown is percent of total thymocytes ± SD. including mouse strain and age. For example, 129/SvPas mice have §Total cell number in thymus of DSS-treated mice declined 57%. low susceptibility, C3H/HeJ have high susceptibility, and C57BL/6

Trottier et al. PNAS Early Edition | 3of6 Downloaded by guest on September 26, 2021 experienced the greatest losses (Table 1). The somewhat greater survival of progenitor and immature-mature B cells might result from increased resistant to cell death. Greater survival of pro-B cells as observed here has also been noted in malnutrition and stress and may allow for rapid regeneration upon normalization of conditions (21). Analogous findings for alterations in lymphopoiesis were noted in the highly atrophied thymuses of DSS- treated mice where the number of developing T cells was de- creased by nearly 60%—verification that colitis significantly alters both primary tissues. As was the case for marrow B cells, there + were substantial losses in the numbers and proportion of CD4 + CD8 pre-T cells (Tables 2 and 3) in colitis mice. On a percentage basis, the depletion of double-negative and single-positive pop- + + ulations was not as extensive as that for CD4 CD8 cells (Table 3). Similar results including thymic atrophy in DSS-induced colitis + have been observed previously, including large losses in CD4 + CD8 thymocytes after treatment of mice with DSS (27). The changes in the proportion and absolute number of T cells and B cells could also be due to reduction in the production of growth factors and cytokines needed to promote their development and differentiation. However, the disproportional losses among pre–T- and pre–B cells, which have low expression of antiapoptotic Fig. 2. Apoptosis of thymocytes from DSS-treated mice. Thymocytes were proteins and are prone to cell death, suggested that enhanced ap- harvested from control and DSS-treated mice and assayed for apoptosis after optosis might be occurring. Indeed, thymocytes from DSS-treated 8 h in culture. Data shown are percent apoptosis of major subpopulations of mice exhibited a 40% increase in the percentage of cells undergoing thymocytes as determined by DAPI staining. Thymocyte subpopulations apoptosis at day 15 (Fig. 2). Over time, this rate of death would were delineated by labeling with anti-CD4 and anti-CD8 antibodies. Each certainly contribute to thymic atrophy and the loss of thymocytes. + + circle represents percent apoptosis for one mouse; bars represent mean Thymic involution and its associated loss of CD4 CD8 thy- apoptosis rate. Controls, n = 8; DSS, n = 9(*P < 0.01). mocytes during colitis have been reported by others (27, 28). In- terestingly, the use of RU486, an inhibitor of the synthesis of glucocorticoids, partially reduced thymic atrophy in DSS-treated mice display an intermediate response to DSS treatment (23, 24). mice, suggesting a role for stress (28). However, in our studies, we Similarly, young mice tend to display a more severe response than did not observe an increase in glucocorticoid levels at 15 d. In a older mice but are a model for IBD effects in growing children. different scenario, memory T cells adoptively transferred to im- Herein, we show that treatment of young C57BL/6 mice with a low munodeficient SCID mice demonstrate a role for memory T cells in dose (1%) of DSS causes gastrointestinal inflammation (colitis) + the continuance of colitis (29). Transferred CD4 CD45RBhi T that significantly altered hematopoietic functions in the marrow cells induced colitis 6 wk later (29). Similar results were obtained by and greatly changed the composition of the BM. using lymphopenic mice. The memory T cells produced G-CSF and Perhaps the most salient finding was the increase from 34% to GM-CSF, which the investigators assumed might account for the + 60% in granulocytes (80% increase) and the 60% increase in greater number of Gr-1 granulocytes found in blood and BM. monocytes in the marrow of DSS mice (Fig. 1). Given the size of Certainly the retention of such T cells from bout to bout of colitis the marrow in the body as a whole, this result represents a large could also extend granulopoiesis and contribute to more intense overall increase in the numbers of cells of the myeloid lineages bouts of colitis. being produced in the body of colitis mice. This change is a clear Understanding the changes created by colitis in the marrow demonstration that colitogenic changes in the body enhance mye- provides opportunities to revise current therapies in favor of lopoiesis, which would contribute significantly to the ongoing tissue fl – more effective ones. Natural and synthetic glucocorticoids are damage and in ammation in the gut (3 5). The larger numbers of the most extensively used drugs for the treatment of UC and CD these cells noted in the peripheral blood suggest that cells of the (30), but they do not prevent future bouts of disease. Moreover, myeloid lineages are moving from the marrow into the periphery it is recognized that prolonged treatment with glucocorticoid can (Table 4). increase opportunistic infections and even lead to organ damage Anemia and loss of weight accompany IBD diseases (1, 2, 17). A (30, 31). The overt increase in myelopoietic cells in the marrow 2011 study indicates that 42% of adults with colitis are anemic (17). noted herein among mice with colitis presents yet another im- – Moreover, 18 62% of individuals with colitis experience weight portant problem. It has been repeatedly shown that glucocorti- loss (25). Bleeding, ulcerative lesions, and bloody diarrhea are as- coids whether provided orally or generated endogenously during sociated with loss of iron and can affect nutrient absorption as well. disease and stress substantially enhance myelopoiesis (8, 10, 32). With these collective changes in iron, vitamins, and other nutrients as a bout of colitis proceeds, it is not surprising that we observed a decline of 40% in the proportion and relative numbers of cells of Table 4. Blood differential counts in control and DSS-treated the erythrocytic lineage in the marrow of DSS-treated mice (Fig. 1). mice Evidence of the onset of anemia is apparent in the periphery where % of white blood cells significantly reduced numbers of red blood cells, blood hemoglobin, and hematocrit are noted (Table S1). Reduced nutriture alone Treatment Neutrophils Lymphocytes Monocytes

can reduce erythrocytic lineages in a manner similar to colitis (9, † ‡ 26) and would contribute to anemia. Control 14.2 ± 3.9 80.0 ± 4.8 3.15 ± 1.7 As is evident, DSS treatment greatly altered lymphopoiesis in the DSS 37.3 ± 11.8** 56.1 ± 12.1** 5.01 ± 4.1 marrow and thymus. In the marrow, lymphoid lineages declined **P < 0.01. 70%, from about 26% to 7.7% of nucleated cells in the BM †n = 6 for controls, n = 8 for DSS. (Fig. 1). The pre-B cells that are prone to undergo apoptosis ‡Data shown is % of white blood cells ± SD.

4of6 | www.pnas.org/cgi/doi/10.1073/pnas.1213854109 Trottier et al. Downloaded by guest on September 26, 2021 Moreover, it has been known for years that promotion of myelo- Cecum Histology. To monitor and confirm colitis induction, a scoring system poiesis in primary BM cultures can readily be accomplished by was developed based on previous reports (33–35) (SI Materials and Methods). adding Gc (32). Furthermore, glucocorticoids are notorious for To further confirm colitis, RNA was extracted from the proximal colon to initiating apoptosis among pre-B cells in marrow and pre-T cells in assess cytokine expression levels as described (11). the thymus (8, 22). Although treatment of UC and CD patients with glucocorticoids may reduce disease symptoms temporarily, BM Harvest and Immunophenotyping. BM from control and DSS-treated mice they may also inadvertently promote even greater myelopoiesis was harvested and processed as described (10, 22). BM cells were labeled with anti–Ly-6C (clone ER-MP20), anti-CD31 (clone ER-MP12; AbD Serotec), and that could prolong a bout of the disease. Additional reductions in fi lymphopoiesis created by these steroids might well increase the anti-Ly-76 (clone Ter119) antibodies (10) to delineate ve major hematopoietic subclasses (SI Materials and Methods). The five populations identified likelihood of opportunistic infection. These changes add to the − − + + were as follows: erythroid (CD31 Ly6-C Ter119 ), lymphoid (CD31 Ly6- growing concerns about the efficacy of the wide use of gluco- − − − + − C Ter119 ), granulocytes (CD31 Ly6-C Ter119 ), heterogeneous monocytes + ++ − + + − corticoids for treatment of IBD (31). (CD31 Ly6-C Ter119 ), and committed progenitors (CD31 Ly6-C Ter119 ) Erythropoietin used in conjunction with iron supplemen- (8, 15). Determination of composition of B lineages in marrow was done as tation could be used to promote erythropoiesis. Interleukin 7, described (26, 36) and is outlined in SI Materials and Methods. which promotes lymphopoiesis among pro- and pre-T and B cells, could be used if its production by stromal cells is compromised. Thymocyte Harvest and Immunophenotyping and Quantitation of Apoptosis. Although often more difficult to implement, antibodies that would Thymuses were processed and labeled as described (37). Briefly, thymocytes neutralize myelopoietic factors, such as granulocyte-macrophage were labeled with anti-CD4 and anti-CD8 (eBiosciences) to determine major − − colony stimulating factor (GM-CSF), might reduce the elevated subsets of thymocytes. For CD4 /CD8 subsets, cells were labeled with anti- production of macrophages and neutrophils. Indeed, a combi- CD25, anti-CD44, c-kit, anti-CD4, and anti-CD8 antibodies. Cells were gated − − + − + nation of these therapies might reduce the length and intensity of on CD4 /CD8 cells, and delineated as follows: DN1, CD44 /CD25 /ckit ;DN2, + + + − − − − + − bouts of colitis, offsetting the need to use glucocorticoids while CD44 /CD25 /ckit ;DN3,CD44/CD25 /ckit ; DN4, CD44 /CD25 /ckit (SI improving patient outcomes. Materials and Methods). Apoptosis of thymocytes was detected flow cyto- In sum, colitis-like conditions substantially altered hematopoi- metrically after culture of isolated cells as described (36, 37) (SI Materials etic processes in the marrow. However, to be identified are po- and Methods). tential changes in cytokine levels, growth factors, and stromal cell activities within the BM that may contribute to the observed Flow Cytometric Analysis. Flow cytometry was performed with a Becton fl fl changes in composition. Similarly, changes in activity of intestinal Dickinson FACS Vantage ow cytometer or a BD LSRII ow cytometer (BD IMMUNOLOGY cells and/or mesenteric lymph nodes undoubtedly will have a great Immunocytometry; DIVA 4.1). Unlabeled samples and single-color labeled impact on the changes occurring both in the bone and the colon. cells served as color controls. Data were analyzed by using WinList software Future analyses need to also identify the sequence of events oc- (Verity Software). curring over time. It would also be important to understand how Blood Analysis. Blood was collected from anesthetized mice by cardiac frequent bouts of colitis might affect hematopoietic function. fi puncture. A complete blood count with differential test was performed by Moreover, it will be interesting to see whether these ndings are the Diagnostic Center for Population and Animal Health at Michigan State reproducible in different rodent models of colitis that work through fl University for analysis of red and white blood cell counts, hematocrit, and other in ammatory responses (Th1 vs. Th2) and in humans. Taken blood hemoglobin, and for differential counts of white blood cells. together our studies contribute to the further understanding of these issues to provide much-needed novel therapies. Statistical Analysis. Data were analyzed by using the Student t test. Statistical significance was set at P < 0.05. Unless otherwise stated, means ± SD are Materials and Methods reported. The number of mice per treatment group was n = 8–11 unless oth- Mouse Model. C57BL/6 males (6 wk old; Harlan Laboratories) were given 1% erwise stated. All mouse experiments were performed two or more times. (wt/vol) DSS (36,000–50,000 MW, catalog no. 160110: MP Biomedical) in sterile water for 15 d to induce colitis. Mice were housed in a 12-h light/dark ACKNOWLEDGMENTS. We thank Afia Naaz for technical assistance. This cycle room at 23 °C and provided standard chow from Teklad. Body weight study was supported by funds provided by the College of Natural Sciences was monitored throughout the study. All protocols were approved by the and AgBioResearch at Michigan State University (P.J.F.) and the Crohn’sand Michigan State University Animal Use Committee. Colitis Foundation of America (L.R.M.).

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