Colonic epithelial response to injury requires Myd88 signaling in myeloid cells

Malvin, N P; Seno, H; Stappenbeck, T S

doi:10.1038/mi.2011.65

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Published: 18 January 2012

Colonic epithelial response to injury requires Myd88 signaling in myeloid cells

N P Malvin^1,2,
H Seno^1,2 &
T S Stappenbeck¹

Mucosal Immunology volume 5, pages 194–206 (2012)Cite this article

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Abstract

Proper colonic injury response requires myeloid-derived cells and Toll-like receptor/Myd88 signaling. However, the precise role of Myd88 signaling specifically in myeloid-derived cells that occurs during tissue damage is unclear. Therefore, we created a mouse line with Myd88 expression restricted to myeloid lineages (Myd88^−/−; LysM^Cre/+; ROSA26^Myd88/+; herein Mlcr). In these mice, Myd88 was appropriately expressed and mediated responses to bacterial ligand exposure in targeted cells. Importantly, the severe colonic epithelial phenotype observed in dextran sodium sulfate-injured Myd88^−/− mice was rescued by the genetic modification of Mlcr mice. During injury, myeloid cell activation and enrichment of Ptsg2-expressing stromal cells occurred within the mesenchyme that surrounded the crypt bases of Mlcr and Myd88^+/− mice but not Myd88^−/− mice. Interestingly, these cellular changes to the crypt base mesenchyme also occurred, but to a lesser extent in uninjured Mlcr mice. These results show that Myd88 expression in myeloid cells was sufficient to rescue intestinal injury responses, and surprisingly, these cells appear to require an additional Myd88-dependent signal from a non-myeloid cell type during homeostasis.

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Introduction

The mouse colon is a distinctive model system that can be utilized to elucidate functional cellular components that mediate damage responses. The epithelium is an absorptive barrier composed of a single layer of cells lining the inner surface of the colonic tube. The apical side of this barrier contacts a substantial and diverse set of indigenous microbes while the basal side contacts cells of the immune system and the stroma.^{1, 2, 3} Studies of colonic wound responses and repair suggest this process requires interaction with both the apically located microbes and the baso-laterally located cells of the innate immune system and stroma.⁴

The mouse colonic epithelium is designed to quickly respond to injury. Cellular turnover is already quite elevated during homeostasis (∼3–5 days). New epithelial cells are supplied from ∼100,000 crypts that are organized in a high-density array (∼400 crypts mm⁻²). Stem and proliferative progenitors are located at the base of each crypt.^{5, 6} Their daughters exit the cell cycle, and migrate upwards and eventually out of crypts onto the inner surface of the intestine. To properly respond to injury, stem and progenitor cells alter their activity to repopulate damaged crypts and the epithelial barrier.⁷

Interaction of the epithelium with mesenchymal cells is critical for proper injury response.^{8, 9} In the colon, a number of cell types have been proposed to have a role in injury repair using a variety of experimental systems.^{2, 3} Myeloid cells are often a prominent cell type within areas of intestinal damage, and engage in an important function of killing and clearing microbes. Under certain circumstances, these cells appear to have a role in the proper response to injury. For example, in the intestine, mice with greatly diminished peripheral myeloid cells (Csf1^op/op mice) when injured with the toxin dextran sodium sulfate (DSS) show loss of proliferation in colonic epithelial progenitors supporting a positive role for myeloid cells in the response to tissue injury.^{10, 11} Elimination of myeloid cells using the inducible diphtheria toxin receptor model showed that these cells have a role in specific phases of skin wound healing.¹²

Toll-like receptor (TLR) signaling through the Myd88 adaptor protein is a critical signaling pathway required for proper colonic injury response to DSS.^{13, 14} This signaling is responsible for microbial recognition, induction of antimicrobial products, and modulation of the adaptive immune response.^{15, 16} TLRs are key recognition molecules within the colon, functioning within many specialized cells, potentially including epithelial cells, myofibroblasts, and professional immune cells.^{17, 18}

We previously found that Myd88 signaling was upstream of focal prostaglandin E2 (PGE2) production during DSS injury.¹³ A rate-limiting enzyme for the production of PGE2 is Ptsg2 (also known as Cox-2).¹⁹ We and others have found that this gene is highly expressed in a population of non-hematopoietic cells in the stroma of the mouse colon,^{13, 20} and that in vitro these cells are consistent with mesenchymal stem cells.²¹ In non-ulcerated areas of DSS damage in the mouse rectum, we found that the positioning of the Ptgs2-expressing stromal cells (PSCs) was important to maintain colonic epithelial proliferation during this injury.¹³ We proposed that the position of these cells was important because PGE2 has a very short half-life and its delivery closer to intestinal epithelial progenitors that are its target would increase its effects.²² PGE2 has been proposed to stimulate Wnt signaling, which is required for intestinal epithelial progenitor function during injury.²³

Our model is that to maintain colonic epithelial proliferation during DSS injury, Myd88 signaling is required in a lineage distinct from the PSCs. One reason is that the latter cell type does not respond to TLR ligands, such as lipopolysaccharide (LPS), as they lack CD14 expression.²¹ Based on our findings with Csf1^op/op mice,¹⁰ we developed the hypothesis that Myd88 signaling was required in myeloid cells for the proper response to DSS damage. To test this hypothesis, we created a novel mouse line with Myd88 expression that was restricted to these cells. Here, we show that our targeting strategy produced appropriate expression of Myd88 in myeloid cells and that this expression was sufficient to rescue the phenotype observed in DSS-injured Myd88^−/− mice.

Results

Generation and validation of the Mlcr mouse strain

We engineered a mouse line that contained Myd88 expression in myeloid-derived cells. We “knocked in” the Myd88 complementary DNA (cDNA) into the Rosa26 locus immediately downstream of its floxed stop signal (Figure 1a–c).^{24, 25, 26} ROSA26^Myd88/+ mice were bred to Myd88^−/− and their progeny were then bred to LysM^Cre/Cre mice. We used this Cre line as recombination has been widely reported to occur in myeloid lineages, including macrophages, GR-1⁺ leukocytes, and dendritic cells.^{27, 28} The breeding scheme generated Mlcr mice (Myd88^−/−;LysM^Cre/+; and ROSA26^Myd88/+) as well as two littermate control groups: Myd88^+/−;LysM^Cre/+; and ROSA26^+/+ (herein Myd88^+/−) and Myd88^−/−; LysM^Cre/+; and ROSA26^+/+ (herein Myd88^−/−). We generated Myd88^+/− mice as controls as mice with this genotype have been shown to be functionally equivalent to Myd88^+/+ mice.²⁹ Using breeding scheme one (generated Mlcr, Myd88^−/−, and Myd88^+/− mice), we found that the distribution of mice obtained was not significantly different than the expected Mendelian ratio (Table 1). Using breeding scheme two (generated Mlcr and Myd88^−/− mice), we found that Mlcr mice were present at ratios significantly higher than the expected Mendelian frequencies (Table 1).

Table 1 Mlcr mice exhibited improved survival as compared with Myd88^−/− littermates

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To validate the genetic targeting, we determined the level of Myd88 expression in myeloid cells and fibroblasts isolated from colons of Mlcr and control mice (Myd88^+/− and Myd88^−/−). We isolated an enriched population of colonic myeloid cells (<95% F4/80 positive) using a protocol based on multiple previous studies^{30, 31, 32, 33} (Supplementary Figure S1 online). Using quantitative quantitative real-time PCR analysis, Myd88 expression was at the limit of detection in myeloid cells isolated from Myd88^−/− mice and was detectable at comparable levels in cells isolated from Mlcr and Myd88^+/− mice (Figure 2a). To confirm the specificity of LysM-Cre in the Mlcr mice, we evaluated Myd88 messenger RNA (mRNA) expression in colonic fibroblasts isolated as previously described.²¹ Myd88 expression was at the limit of detection in fibroblasts isolated from Mlcr and Myd88^−/− mice, and was detectable in cells isolated from Myd88^+/− mice (Figure 2b). We confirmed in separate experiments that Myd88^+/+ and Myd88^+/− mice from our colony expressed similar levels of Myd88 mRNA (Supplementary Figure S2 online). We also performed immunofluorescence localization of Myd88 on colonic sections using tyramide amplification. By this method, we detected Myd88 in the crypt epithelium of Myd88^+/+ and Myd88^+/− mice but not in the epithelium of Mlcr and Myd88^−/− mice (Supplementary Figure S3 online). Thus, constitutive expression of Myd88 mRNA occurred as expected based on the known cellular targets of LysM-Cre.

Myeloid cells in Mlcr mice have functional expression of Myd88

To perform functional tests of Myd88 expression in Mlcr mice, we isolated bone marrow monocytes and performed in vitro differentiation for macrophages (M-CSF) and dendritic cells (GM-CSF),³⁴ and confirmed appropriate expression of Myd88. We chose this model as both of these cell populations are readily expanded in vitro. As expected, Myd88 expression in macrophages and dendritic cells derived from Myd88^−/− mice was at the limits of detection. Myd88^+/− and Mlcr mice expressed comparable levels of Myd88 mRNA in bone marrow-derived macrophages (Figure 3a). As an additional control, we found the levels of Myd88 expression were similar in Myd88^+/− and Myd88^+/+ bone marrow-derived macrophages (Supplementary Figure S4a online). The levels of Myd88 expression in dendritic cells from Mlcr mice were 1.6-fold less than those isolated from Myd88^+/−.

As Myd88 expression is critical to drive responses to bacterial products,^{14, 15, 16} we treated bone marrow-derived macrophages and dendritic cells from Mlcr and control mice with LPS, and evaluated gene expression of two well-known targets, Ptgs2 and tumor necrosis factor (TNF)-α.^{15, 21} At baseline, gene expression for both Ptgs2 and TNF-α showed no significant differences based on genotype for both macrophages and dendritic cells (Figure 3b and c; Supplementary Figure S4b, c online). LPS-treated macrophages and dendritic cells from Myd88^+/− and Mlcr mice expressed higher levels of Ptgs2 and TNF-α as compared with corresponding LPS-treated Myd88^−/− cells (Figure 3d and e; Supplementary Figure S4b, c online). Taken together, these results were consistent with functional levels of Myd88 expression in myeloid cells isolated from Mlcr mice.

Myeloid expression of Myd88 rescues the DSS injury phenotype of Myd88^−/− mice

We next evaluated the colonic damage response of adult mice treated for 1 week with 2.5% DSS. This experimental treatment created multiple focal ulcers in the transverse colon as well as a single confluent ulcer at the ano-rectal junction in Mlcr mice as well as their littermate controls. Importantly, the descending colons of DSS-treated Mlcr mice and controls were neither ulcerated nor inflamed as evidenced by the absence of accumulated mucosal neutrophils in this location for any group of DSS-treated mice (Supplementary Figure S5 online). These findings were similar to previous studies with DSS-treated mice.^{10, 13}

We next performed histological analysis of descending colons from groups of untreated and DSS-treated mice. The descending colon of untreated Mlcr mice was similar in appearance to untreated colons from Myd88^+/− and Myd88^−/− mice (Figure 4a–c). We found no discernable differences when comparing untreated and DSS-treated Myd88^+/− mice (Figure 4a and d). In contrast, DSS-treated Myd88^−/− mice contained extensive crypt atrophy, loss of crypt epithelial cells, and a thickened muscularis propria as compared with untreated Myd88^−/− mice (Figure 4b and e). Interestingly, the descending colons from DSS-treated Mlcr mice showed no apparent differences in comparison with untreated Mlcr mice (Figure 4c and f). These results showed that expression of Myd88 in myeloid cells rescued the effects of DSS injury in Myd88^−/− mice. LysM targets multiple myeloid cell types including macrophages, dendritic cells, and neutrophils.^{27, 28} We found that ablation of neutrophils in wild-type mice did not alter the epithelial response to DSS treatment, suggesting this cell type is not required (Supplementary Figure S6 online).

As the descending colonic crypts were a primary morphological target of DSS in Myd88^−/− mice, we qualitatively evaluated crypt size by H+E staining and epithelial proliferation by 5-bromo-2-deoxyuridine incorporation to visualize cells in S-phase (Figure 5a–f). These stained sections showed that crypts from DSS-treated Myd88^−/− mice contained fewer total and S-phase cells as compared with all other groups of mice. We next performed quantitative analysis of epithelial proliferation, cell census, and cell death of the intestinal epithelium in DSS-treated and untreated mice (Figure 5g–j). Quantification of total numbers of epithelial cells/crypt, M-phase cells/crypt, and S-phase cells/crypt showed that DSS-treated Myd88^−/− mice had a significant reduction in cell census and proliferation as compared with untreated Myd88^−/− mice (Figure 5g–i). These differences were present for all the genetic subgroups of Myd88^−/− mice obtained by our breeding scheme (Supplementary Figure S7 online). Importantly, we found that DSS-treated Mlcr mice showed no reduction in epithelial cell census and proliferation compared with untreated Mlcr mice (Figure 5g–i). The findings for DSS-treated Mlcr mice were comparable to similarly treated Myd88^+/− controls (including subgroups of Myd88^+/−, Supplementary Figure S7 online). Quantification of apoptosis was determined by visualization of apoptotic bodies in the epithelium.^{10, 13} This analysis showed no significant differences when comparing DSS-treated and untreated mice for all three genotypes (Figure 5j).

Mlcr mice contain activated colonic myeloid cells during DSS injury

We previously found that based on morphology, activated myeloid cells were present in the peri-crypt base mesenchyme of DSS-treated wild-type but not similarly treated Myd88^−/− mice.¹⁰ To test if Myd88 expression within myeloid cells stimulated activation during injury, we analyzed slides co-labeled with antisera directed against F4/80 and CD86. The latter is a recently established marker of macrophage activation.^{35, 36, 37, 38} In mice of all three genotypes, CD86-positive aggregates were preferentially located in the crypt base-associated mesenchyme and often co-localized with F4/80 (Figure 6a and b). Quantification of the number of double-positive aggregates (CD86 and F4/80) per crypt-associated mesenchyme showed significantly higher numbers in DSS-treated Myd88^+/− and Mlcr mice as compared with untreated genotype controls (Figure 6c). As an additional control, the numbers of double-positive aggregates were similar in DSS-treated and untreated Myd88^+/− and Myd88^+/+ (Supplementary Figure S8 online). In contrast, the number of double-positive aggregates was not significantly increased in DSS-treated vs. untreated Myd88^−/− mice (Figure 6c). As an additional control, we found that nearly all of the CD86-positive aggregates co-localized with F4/80 in DSS-treated Myd88^+/−, Myd88^+/+, and Mlcr mice (Figure 6d; Supplementary Figure S8 online). These data support a major role for Myd88 signaling in myeloid cell activation during DSS injury.

Mlcr mice contain PSCs that are preferentially distributed near the crypt base

We previously found that in response to DSS treatment, PGE2 produced by the rate limiting enzyme Ptsg2 acts downstream of Myd88 in order to maintain colonic epithelial proliferation.²¹ We also previously established that Ptgs2 expression is readily detectable in mesenchymal stem cells (a.k.a. fibroblasts) both in vivo and in vitro.²¹ Interestingly, it is the distribution of PSCs that is dependent on Myd88 signaling and is associated with maintenance of colonic epithelial proliferation during DSS treatment.¹³

Therefore, we determined the localization of Ptgs2 in tissue sections from DSS-treated and -untreated mice from all three genotypes. In all groups, we observed scattered PSCs that were negative for F4/80 and CD11c antigens (Figure 7a–f). These cells were consistent with fibroblasts by additional marker studies.¹³ Quantification of PSCs showed no significant differences in abundance of these cells when comparing groups based on genotype or treatment (Figure 7g), similar to previous studies.¹³ We then determined the levels of Ptgs2 mRNA expression in colonic fibroblasts and colonic myeloid cells isolated from Mlcr, Myd88^+/−, and Myd88^−/− mice. For all three genotypes, we found that the expression of Ptsg2 in fibroblasts was significantly higher than myeloid cells (Figure 7h). Additional experiments showed Myd88^+/+ also expressed higher levels of Ptgs2 in fibroblasts as compared with myeloid cells (Supplementary Figure S9 online). These findings are consistent with previous studies that show PSCs (fibroblasts) are a prominent source of Ptgs2 in the descending colon in both untreated and DSS-treated mice of multiple genotypes.^{13, 20}

We previously found that the localization of PSCs was altered in response to DSS treatment in a Myd88-dependent fashion.¹³ As anticipated, PSCs were enriched in the peri-crypt base region of DSS-treated Myd88^+/− mice (vs. untreated controls) but were not enriched in this region of DSS-treated Myd88^−/− mice (vs. untreated controls; Figure 8a and b). As an additional control, we compared the distribution of PSCs in untreated and treated Myd88^+/− vs. untreated and treated Myd88^+/+ (that we previously reported)¹³ and found no differences. Importantly, DSS-treated Mlcr mice contained a similar enrichment of PSCs in the peri-crypt base region (vs. untreated Mlcr mice). Thus, expression of Myd88 in myeloid cells was sufficient to alter the distribution of PSCs during DSS injury.

Myeloid cells in untreated Mlcr mice are in a mildly activated state

An unexpected finding was an enrichment of PSCs in the peri-crypt base region of untreated Mlcr mice as compared with untreated Myd88^+/− and Myd88^−/− mice (Figure 8b). As the DSS experiments showed that myeloid cell activation was correlated with altered PSC distribution, we reanalyzed the data from Figure 6c considering only the untreated groups of mice and found a significantly greater number of double-positive (CD86/F4/80) aggregates in untreated Mlcr mice as compared with both untreated control groups (analysis of variance with a Tukey's post-test, P<0.01). This finding indicated a mild level of colonic myeloid cell activation in untreated Mlcr mice. We then evaluated mRNA expression of additional markers of myeloid cell activation and found small but significant increases in isolated colonic myeloid cells from untreated Mlcr mice as compared with control groups (Figure 9; Supplementary Figure S10 online). This difference was not correlated with an alteration in the expression of anti-inflammatory cytokines in Mlcr mice (Supplementary Figure S11 online). Thus, the myeloid cells in Mlcr colons appear to be mildly activated.

As enhanced activation of NFκB in the intestinal epithelium leads to inflammation, fibrosis, and tumorigenesis in aged mice,³⁹ we evaluated the gross and histological pathology of 12–13-month-old Mlcr mice. Interestingly, there was no evidence of inflammation, fibrosis, or tumorigenesis. In contrast, there was a decrease in epithelial proliferation in the transverse colon as compared with littermate controls (Supplementary Figure S12 online).

Discussion

In this study, we determined the role of Myd88 signaling in myeloid-derived cells. We first created a mouse model (Mlcr) where Myd88 expression was directed to this lineage using a myeloid-specific Cre and a Myd88 cDNA knocked into the ROSA26 locus. We found this genetic system appropriately restricted Myd88 expression to cell types known to be targeted by LysM-Cre and mediated effects in isolated myeloid-derived cells. Importantly, we showed that restricted expression of Myd88 using LysM-Cre was sufficient to rescue the phenotype in Myd88^−/− DSS-injured mice. The colonic features of this mouse model during DSS treatment were comparable to similarly treated Myd88^+/− mice, and included epithelial responses to damage, myeloid cell activation, and localization of PSCs. Interestingly, we found that in uninjured mice, the Mlcr line contained mildly activated colonic myeloid cells, indicating that Myd88 signaling in non-myeloid lineages can inhibit myeloid activation.

Myd88 has a role in numerous biological processes within a variety of tissues that include wound repair in lung⁴⁰ and skin⁴¹, cancer⁴² and host defense against pathogens.⁴³ Creation of a floxed allele of this gene by other investigators has been beneficial to determine if deficiency in a particular lineage(s) is required for the function of Myd88 in the whole animal.^{44, 45} In contrast to a transgenic over-expression approach,⁴⁶ we designed our model with the intention to create a flexible genetic system based on breeding to additional Cre lines. In addition, we found that Mlcr mice contained levels of Myd88 expression in myeloid cells that was comparable to myeloid cells isolated from Myd88^+/− and Myd88^+/+ mice. In contrast to bone marrow chimeras^{47, 48} where radio-resistant tissue myeloid cells could possibly confound interpretation, our system has the advantage of more precise control of Myd88 expression. We anticipate that our model should complement findings using a floxed Myd88 allele.

We chose LysM-Cre to test the role of Myd88 in myeloid cells as this Cre targets macrophages and dendritic cells^{27, 28} and thus mimics the cell types in the colon that are diminished in mice with Csf1-inactivating mutations. Thus, we could evaluate the role of Myd88 expression in comparable cell types. LysM-Cre also targets neutrophils.^{27, 28} Here and in our previous study, we have found no obvious role for neutrophils in the response to DSS injury.¹⁰ In addition, we examined an area of the colon that does not undergo extensive damage during DSS treatment and thus there is no accumulation of neutrophils in this area. For these reasons, we focused our analysis on monoctye-derived cells in Mlcr mice.

We previously found a robust, Myd88-dependent, simultaneous elevation of mRNA signatures for both classical and alternative forms of macrophage activation in the mesenchyme of DSS-treated mice.¹⁰ We evaluated the expression of CD86 as it is well recognized as a marker of activated macrophages under both conditions of classical and alternative activation. Elevated CD86 expression in myeloid cells has been reported in a model of spontaneous colitis (Stat3^f/f;LysM^Cre/+).⁴⁹ CD86 can be expressed in antigen-presenting cells where it has co-stimulatory properties.^{35, 38, 50} Here, using CD86 as a readout, we found that Myd88 expression in myeloid cells was sufficient to drive myeloid cell activation during DSS treatment.

An unexpected finding was that colonic myeloid cells in untreated Mlcr mice showed evidence of mild activation as compared with untreated Myd88^+/− mice. This finding suggests that a source of Myd88 stimulation was unmasked and could include either microbial- or host-derived factors. The finding also suggests the possibility that an additional cellular source of the Myd88 expression may reduce myeloid activation in a non-cell-autonomous manner. This question is of importance as myeloid-derived cells in the intestine are hypoactive in the gut.^{2, 3} Thus, the Mlcr model should be an interesting system to approach this question and determine the interactive roles of Myd88 signaling in multiple lineages of the intestine.

Materials and Methods

Mice. The Washington University Medical School Animal Studies Committee approved all animal experiments. Mice were maintained in a specific pathogen-free barrier facility under a strict 12-hour light/dark cycle and fed autoclaved chow diet (B&K Universal, Grimston, Aldbrough, UK). The 6–10-week-old mice were used for experiments. We treated with DSS as previously described.^{10, 13}

Generation of Mlcr mice. We subcloned the cDNA for Myd88 into Rosa26PA.^{24, 25} The linearized targeting vector was electroporated into F1 B6/129SV ES cells. Positive clones were identified by Southern blotting, injected into C57/B6 blastocysts, and implanted into surrogates. Mice with germline transmission were bred to Myd88^−/−, ^{10, 13} and LysM^Cre/Cre mice (Jackson Laboratories, Bar Harbor, ME). All experimental mice were littermates on a mixed C57B6/129SV background.

Genotyping. Myd88 locus. Primers: Myd88 Forward 5′-TGG CAT GCC TCC ATC ATA GTT AAC C-3′; Myd88; Reverse 5′-GTC AGA AAC AAC CAC CAC CAT GC-3′; Myd88 Neo 5′-ATC GCC TTC TAT CGC CTT CTT GAC G-3′.

PCR protocol: 5 °C for 2 min; followed by 30 cycles of: 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; followed by 72 °C for 5 min. PCR products were visualized on 2.75% TBE agarose gels run for 1.5 h at 100 V to clearly separate the amplicons. A 550 base-pair amplicon indicated a wild-type Myd88 locus and a 600 base-pair amplicon indicated a knockout Myd88 locus.

Mlcr locus. Primers: Forward 5′-CCC CAA CGA TAT CGA GTT TG-3′; Reverse 5′-CTT GGT GCA AGG GTT AT-3′.

PCR: 95 °C for 2 min; followed by 35 cycles of: 95 °C for 20 s, 55 °C for 30 s, and 72 °C for 70 s; followed by 72 °C for 5 min. Refer to Table 2 for interpretation of the three potential amplicons.

Table 2 Interpretation of Mlcr genotyping

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LysM-Cre locus. Primers: Forward 5′-AGG TTC GTT CAC TCA TGG A-3′; Reverse 5′-TCG ACC AGT TTA GTT ACC C-3′.

PCR: 95 °C for 2 min; followed by 30 cycles of: 95 °C for 30 s, 60 °C for 30 s, and 72 °C for 30 s; followed by 72 °C for 5 min. A 220 base-pair amplicon indicated presence of Cre in the LysM locus.

Histochemistry and immunohistochemistry. Colons were dissected, submerged in Bouin's fixative for 4 h at 24 °C or methacarn fixative for 2 h at 24 °C, and embedded in 2% agar for paraffin processing. Serial 5-μm-thick sections of the descending colons were cut along the cephalocaudal axis and perpendicular to the mucosal surface. Sections were stained with hematoxylin and eosin.¹⁰ For frozen sections, dissected colons were flushed first with phosphate-buffered saline (PBS) followed by OCT media, arranged as 2 cm segments in histology cassettes and frozen with cytocool. Serial 7-μm-thick sections of descending colons were cut along the cephalocaudal axis and perpendicular to the mucosal surface.

We performed intraperitoneal injections in mice with a solution containing 5-bromo-2-deoxyuridine (120 mg kg⁻¹) and 5′-fluor-2′-deoxyuridine (12 mg ml⁻¹; Sigma-Aldrich, St Louis, MO) 1 h before killing. S-phase cells were identified in tissue sections using goat anti-5-bromo-2-deoxyuridine IgG.^{10, 13}

Ptgs2 localization was performed on 5-μm-thick sections of methacarn-fixed tissues stained with mouse IgG1 anti-Ptgs2 mAb (clone 33; BD Biosciences, San Diego, CA; 1:50) and the Zenon antibody labeling system (Invitrogen, Grand Island, NY) as previously described.¹³ For co-labeling experiments with Ptgs2, F4/80, and CD11c, 7-μm-thick sections of frozen tissue were stained with mouse IgG1 anti-Ptgs2 mAb (clone 33; BD Biosciences; 1:50) and the Zenon antibody labeling system (Invitrogen), rat IgG2b anti-mouse F4/80 mAb directly conjugated to Alexa-488 (clone CI:A3-1; Biolegend, San Diego, CA; 1:100) and Armenian hamster IgG anti-mouse CD11C mAb directly conjugated to Alexa-647 (clone N418; Biolegend; 1:50 O/N 4 °C). Ig isotype controls for all antibodies used for all immunofluoresence studies. Myd88 was detected on frozen, methanol-fixed sections using an overnight incubation of rabbit anti mouse Myd88 antisera (Abcam, Cambridge, MA; 1:750 dilution) followed by tyramide amplification of signal (Invitrogen).

Co-localization of the macrophage activation marker CD86 and F4/80 was performed on 7-μm-thick sections of frozen tissue stained with rat IgG2a anti-mouse CD86 mAb directly conjugated to PerCP (clone GL:1; Biolegend; 1:100) and rat IgG2b anti-mouse F4/80 mAb directly conjugated to Alexa-488 (clone CI:A3-1; Biolegend; 1:100). These stained tissue sections were used to analyze CD86 aggregate per crypt base mesenchyme that co-localized with F4/80-positive cells.

Cell culture media. Bone marrow-derived macrophage-conditioned media (BM20) consisted of Dulbecco's modified Eagle's medium (DMEM) media supplemented with 10% fetal bovine serum, 20% L-cell supernatant (from L929 fibroblasts), 5% horse serum, 1% L-glutamine, 1% sodium pyruvate, 1:100 of 1 M Hepes, 1 μl ml⁻¹ of 10,000 U ml⁻¹ penicillin/streptomycin (Abx), with media changed on day 4 and 7. Bone marrow-derived dendritic cells culture media (DC10) was RPMI1640 media supplemented with 10% fetal bovine serum, 1 μl ml⁻¹ of 10,000 U ml⁻¹ penicillin/streptomycin, 2% L-glutamine, 2% GM-CSF supernatant (B78hi hybridoma), 1% sodium pyruvate, 1% nonessential amino acids, media changed on day 4, 6, and 7.

Fibroblast media consisted of DMEM media supplemented with 10 mM Hepes, 10% fetal bovine serum, and 1 μl ml⁻¹ of 10,000 U ml⁻¹ penicillin/streptomycin.

Colonic myeloid cell isolation media consisted of DMEM media supplemented with 10 mM Hepes, 1 μl ml⁻¹ of 10,000 U ml⁻¹ penicillin/streptomycin, L-glutamate in both DM10 and DM-Abx, and 10% fetal bovine serum in DM10 only.

Isolation of fibroblasts and myeloid cells from the colon and macrophages, and dendritic cells derived from bone marrow.

Colonic stromal fibroblasts were isolated from individual mice as previously described.²¹ Cells were cultured in supplemented DMEM until confluent, media changed every 3 days.

Bone marrow-derived macrophages and dendritic cells were cultured from bone marrow flushed from the femurs and tibias of a single mouse into 30 ml of DMEM. The cell suspension was split into two equal portions and 35 ml of a specific conditioned media was added to each aliquot. The cell suspensions were then pipetted into 10 × 15-cm non-tissue culture Petri dishes (5 plates, 10 ml per plate), and the cells were incubated in a standard tissue culture incubator for 7 days to stimulate differentiation into either macrophages (incubated in BM20 media) or dendritic cells (incubated in DC10 media). For LPS experiments, cells were treated will EDTA to remove them from the plate. The cells were then replated at 1.25 × 10⁵ cells per ml for 18 h overnight, and then treated for 1 h with 10 ng ml⁻¹ LPS from E. coli (Sigma-Aldrich) or fresh conditioned media.

Mouse colonic myeloid cells were isolated using a protocol derived from multiple previous studies.^{30, 31, 32, 51, 52, 53} Briefly, whole colons were removed, flushed with 1:1 DMEM:PBS, cut into 1 mm pieces using with a sterile razor blade, and the fragments were then placed in a sterile beaker with 40 ml of DM10, 400 μl of 0.5 M EDTA, and 37.7 μl of 1 M dithiothreitol. The suspension was placed in an orbital shaker (250 r.p.m.) for 20 min at 37 °C and then filtered using a 100 μm cell filter (Fisher, Pittsburgh, PA). The tissue fragments were washed three times with shaking in DM-Abx supplemented with 400 μl of 0.5 M EDTA and filtered after each wash. The washed tissue fragments were further minced with sterile razor blade and the fragments were placed in 15 ml of DM-Abx supplemented with 475 μl of 5 mg ml⁻¹ collagenase IV and 150 μl of 10 mg ml⁻¹ DNase. The suspension was placed in an orbital shaker (250 r.p.m.) for 30 min at 37 °C. The suspension was then passed through a 70 μm cell filter. The media were retained and 15 ml of DM10 supplemented with 150 μl of DNase (10 mg ml⁻¹) was added. The suspension was centrifuged at 1,200 g for 5 min at 24 °C. The media was carefully removed and the cell pellet was resuspended in 12 ml of DM10. The cells were then plated in a non-tissue culture-treated Petri dish for 1 h. The adherent cells were washed three times with sterile PBS. The cells were then treated with 5 ml of EDTA/PBS and incubated at 4 °C for 10 min. The plate was scraped to remove adherent cells. The cells were resuspended in 5 ml of DM10 and 5 ml of EDTA/PBS, and centrifuged at 1,200 g for 5 min to pellet the myeloid cells.

RNA extraction and quantitative real-time PCR analysis.

RNA was extracted using NucleoSpin RNA II (Clontech Laboratories, Mountain View, CA) for colonic fibroblasts and bone marrow-derived macrophages and dendritic cells. Colonic myeloid cell RNA was isolated using the PicoPure RNA Isolation Kit (Applied Biosystems, Carlsbad, CA). The quality and quantity of all RNAs were evaluated by Nanodrop (Thermo Scientific, Waltham, MA). Random primed cDNA (Superscript III, Invitrogen) was used as a template for quantitative real-time PCR that was performed in triplicate for each biologic sample using SYBR Advantage (Clonetech, Mountain View, CA). The threshold cycle (C_T) of the gene of interest was subtracted from the baseline C_T (18s mRNA) to calculate a change in C_T (ΔC_T). To calculate the fold change in gene expression, the ΔC_T for each measurement was subtracted from the baseline ΔC_T (average ΔC_T of Myd88^+/− untreated cells mRNA).

Primers used for qPCR.

18S (5′-CATTCGAACGTCTGCCCTATC-3′ and 5′-CCTGTGCCTTCCTTGGA-3′), Ptgs2 (5′-TGCCTGGTCTGATGATGTATG-3′ and 5′-GGGGTGCCAGTGATAGAGTG-3′), TNF-α (5′-CCCTCACACTCAGATCATCTTCT-3′ and 5′-GCTACGACGTGGGCTACAG-3′), and Myd88 (5′-CACCTGTGTCTGGTCCATTG-3′ and 5′-CTGTTGGACACCTGGAGACA-3′). The latter primers were designed to include exons 4 and 5 so as to amplify only intact mRNA as found in Myd88^+/− but not Myd88^−/− mice.

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Acknowledgements

We thank E Unanue, W Stenson, N Manieri, and M Walker for comments on the manuscript, and M White for microinjection of targeted ES cells. This work was supported by the National Institutes of Health (DK071619), Crohn's Colitis Foundation of America, Pew Scholars Foundation, and Washington University Digestive Disease Research Core (NIH P30-DK52574).

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Department of Pathology and Immunology, Washington University School of Medicine, St Louis, Missouri, USA
N P Malvin, H Seno & T S Stappenbeck
These authors contributed equally to this work,
N P Malvin & H Seno

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N P Malvin
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H Seno
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T S Stappenbeck
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Correspondence to T S Stappenbeck.

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Malvin, N., Seno, H. & Stappenbeck, T. Colonic epithelial response to injury requires Myd88 signaling in myeloid cells. Mucosal Immunol 5, 194–206 (2012). https://doi.org/10.1038/mi.2011.65

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Received: 03 August 2011
Accepted: 05 December 2011
Published: 18 January 2012
Issue Date: March 2012
DOI: https://doi.org/10.1038/mi.2011.65

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