CX3CR1 regulates intestinal macrophage homeostasis, bacterial translocation, and colitogenic Th17 responses in mice (original) (raw)
Murine intestinal LP macrophage subsets preferentially express CX3CR1. In the intestine, LP DCs and macrophages play a central role in regulating innate and adaptive immune responses as well as inflammation. These various functions are carried out by different subsets of LP DCs and macrophages. Based on the expression of CD11b, CD11c, F4/80, and CD103, we have identified 4 populations of murine intestinal LP APCs, as defined as CD45+MHC II+ cells (Figure 1A and ref. 18): CD11c+CD11b–CD103+F4/80– DCs (R1), CD11c–CD11b+CD103–F4/80+ macrophages (R3), CD11c+CD11b+CD103+F4/80– DCs (R4), and CD11c+CD11b+CD103–F4/80+ macrophages (R5). For simplicity, we refer to these populations as CD11b– DCs, CD11c– macrophages, CD11b+ DCs, and CD11c+ macrophages, respectively. It is reported that CD11b+ LP DCs extend dendrites between neighboring IECs in order to sample luminal antigens in a CX3CR1-dependent manner (12, 19–21). However, CD11b+ LP cells include a mixture of DC and macrophage subsets, and it is not clear which subsets express CX3CR1. Using Cx3cr1gfp/gfp reporter mice, which have Cx3cr1 replaced with gfp in both alleles, we analyzed the CX3CR1 expression on each of the LP macrophage and DC subpopulations. As shown in Figure 1B, we observed that GFP (CX3CR1) is expressed by CD11c– and CD11c+ macrophages, but not by CD11b– or CD11b+ DCs, in both the small and large intestine. In order to rule out any possibility that GFP is not faithfully marking CX3CR1-expressing cells in this mouse model, we analyzed CX3CR1 expression in Cx3cr1+/+ C57BL/6 mice, using anti-CX3CR1 antibodies. Our results confirmed the GFP expression pattern, with specific CX3CR1 expression by CD11c– and CD11c+ macrophages, but not CD11b– or CD11b+ DCs (Supplemental Figure 1). We also performed quantitative RT-PCR (qRT-PCR) on sorted CD11c– or CD11c+ macrophages, as well as CD11b– or CD11b+ DCs. This further confirmed highly specific expression of CX3CR1 in both macrophages populations, but not in DC subsets (Figure 1C).
Murine intestinal LP macrophages express CX3CR1. (A) Flow cytometry of small (SI) and large (LI) intestine LP cells from C57BL/6 mice, stained for CD45, I-Ab, CD11b, CD11c, F4/80, and CD103. Populations were defined as R1, CD11b– DCs; R3, CD11c– macrophages; R4, CD11b+ DCs; and R5, CD11c+ macrophages. (B) GFP expression in LP macrophages (MΦ; solid histograms) and DCs (open histograms) in the small and large intestine of Cx3cr1gfp/gfp mice, assessed by flow cytometry and gated as in A. (C) qRT-PCR for Cx3cr1 mRNA expression in isolated intestinal LP APCs from Cx3cr1+/+ mice. Data are representative of 3 (B and C) or more than 3 (A) independent experiments. Error bars represent SD. *P < 0.05 versus respective controls.
Reduction of LP macrophages in CX3CR1- and CX3CL1-deficient mice. Having observed specific expression of CX3CR1 by LP macrophages, we performed experiments to investigate the functional role of CX3CR1 in the intestine. Interestingly, phenotypic characterization of LP APCs in Cx3cr1gfp/gfp mice revealed a significant reduction in the frequency of CD11c– and CD11c+ macrophages in Cx3cr1gfp/gfp mice when compared with Cx3cr1+/+ mice in both the small and large intestine (Figure 2A). This reduction was specific for LP macrophage subsets, as no significant differences were observed in the frequencies of CD11b– or CD11b+ LP DCs between Cx3cr1gfp/gfp mice and Cx3cr1+/+ mice. The total cell numbers of the CD11c– and CD11c+ macrophage subsets were also significantly reduced in Cx3cr1gfp/gfp mice, while DC subsets remained largely unaffected (Figure 2B). Since both LP macrophage subsets expressed high levels of GFP and were reduced in number, we wanted to verify that the observed reduction in LP macrophages was the result of CX3CR1 deficiency and not GFP-induced cytotoxicity. Therefore, we performed the same phenotypic characterization of LP DCs and macrophages in a different CX3CR1-deficient mouse line (22) that does not express GFP. Results using these Cx3cr1–/– mice confirmed our observations with Cx3cr1gfp/gfp mice, demonstrating a specific reduction in the frequency (Supplemental Figure 2A) and total cell number (Supplemental Figure 2B) of CD11c– and CD11c+ LP macrophages, but not CD11b– or CD11b+ LP DCs. These data formally exclude GFP-induced cytotoxicity as the explanation for specific LP macrophage reduction in Cx3cr1gfp/gfp mice. Additionally, mice deficient in CX3CL1 (the ligand for CX3CR1) showed a reduction in LP macrophage subsets similar to that observed in CX3CR1-deficient mice (Supplemental Figure 3).
Cx3cr1gfp/gfp mice harbor a specific reduction in LP macrophages. (A) Percentage of CD45+MHC II+ macrophages and DCs in the small and large intestine of Cx3cr1gfp/gfp or Cx3cr1+/+ mice. (B) Total cellularity of CD45+MHC II+ macrophages and DCs in the small and large intestine of Cx3cr1gfp/gfp or Cx3cr1+/+ mice. (C) Percentage of bone marrow MDPs and blood monocytes of Cx3cr1gfp/gfp or Cx3cr1+/+ mice. Data are representative of 3 (C) or more than 3 (A and B) independent experiments. Error bars represent SEM. *P < 0.05 versus respective controls.
To evaluate whether the LP macrophage reduction in Cx3cr1gfp/gfp mice is reflective of a general defect in monocyte/macrophage development or precursor accumulation, we analyzed the frequency of monocyte-DC common precursors (MDPs) in bone marrow as well as circulating blood monocytes (Figure 2C) and did not observe any significant difference in either of these populations. The significant LP macrophage reduction in CX3CR1-deficient mice was also not likely the result of increased cell death, since transfer of CD45.2+Cx3cr1+/+ or CD45.2+Cx3cr1gfp/gfp bone marrow–derived macrophages (BMDMs) into CD45.1+ hosts resulted in equivalent numbers of Cx3cr1+/+ and Cx3cr1gfp/gfp BMDMs in the spleens, but a significant reduction in Cx3cr1gfp/gfp BMDMs in the intestines of recipient mice (data not shown). Taken together, our results demonstrate a specific reduction in LP macrophages in CX3CR1-deficient mice and suggest a role for the CX3CR1/CX3CL1 axis in regulating LP macrophage migration and/or retention in the intestine. Interestingly, CX3CR1 deficiency also resulted in a significant reduction in macrophages in the lung and liver, but not the spleen (Supplemental Figure 4).
Increased bacterial translocation in CX3CR1- and CX3CL1-deficient mice. Given the phagocytic nature of LP macrophages (23) and their significant reduction in Cx3cr1gfp/gfp and Cx3cr1–/– mice, we hypothesized that there may be a defect in the clearance of commensal bacteria that gain access to the lamina propria. To test this, we analyzed the mLNs from these mice for signs of bacterial translocation. Total mLN cell suspensions cultured under aerobic conditions on LB blood agar plates revealed the presence of large numbers of bacteria in Cx3cr1gfp/gfp and Cx3cr1–/– mice (Figure 3, A and B) and in Cx3cl1–/– mice (Supplemental Figure 5A). In most experiments, bacteria could not be grown from mLNs of Cx3cr1+/+ mice. The increased bacteria in the mLNs of CX3CR1-deficient mice was not the result of defective intestinal barrier function, since there was no difference in the uptake of orally delivered, fluorescent 4-kDa and 10-kDa dextran into the serum of Cx3cr1+/+ and CX3CR1-deficient mice (data not shown). Additionally, Cx3cr1+/+ and CX3CR1-deficient macrophages expressed similarly low to undetectable mRNA levels for molecules involved in forming adherens junctions (E-cadherin and β-catenin) and tight junctions (ZO-1, occludin, claudin-1; data not shown); thus, unlike what has been shown for DCs (24), macrophages may not play a role in directly maintaining intestinal epithelial barrier via tight junction protein expression.
CX3CR1-deficient mice have increased bacterial translocation. CFU per mouse in the mLNs of Cx3cr1gfp/gfp (A) and Cx3cr1–/– (B) mice. Relative abundance of phyla (C) and genera (D) among the translocated bacteria. Data are representative of 3 independent experiments, with 4 mice per group per experiment. *P < 0.05 versus respective controls.
To assess whether the bacteria present in the mLNs of CX3CR1-deficient mice that grew under aerobic culture conditions were predominantly commensal or pathogenic bacteria, we isolated DNA from 50 individual colonies and performed PCR amplification using consensus 16S rDNA primers. Sequencing of PCR amplicons revealed that the translocated bacteria were predominantly commensal bacteria corresponding to the phylum Firmicutes (Figure 3C), with Enterococcus being the predominant genus (Figure 3D). Notably, a few sequences corresponded to Shigella; therefore, we cannot exclude the possibility that pathogenic bacteria can also gain access to the LP in the absence of CX3CR1. Overall, these results suggest that CX3CR1-expressing LP macrophages play a vital role in restricting translocation of commensal bacteria to the mLN during steady-state conditions.
Absence of CX3CR1 or CX3CL1 leads to enhanced intestinal inflammation. It has been shown that LP macrophages are important regulators of immune responses during intestinal inflammation (25, 26). Therefore, the specific reduction in LP macrophages in CX3CR1 deficiency prompted us to analyze how these mice respond to acute colitis induced by DSS. Beginning as early as 2 days following DSS administration, Cx3cr1gfp/gfp and Cx3cr1–/– mice exhibited significantly enhanced signs of intestinal disease when compared with Cx3cr1+/+ mice, as defined by increases in overall disease activity index (DAI), a measure of weight loss, fecal blood, and soft stool/diarrhea (Figure 4A). Histological analysis showed greater tissue damage and inflammatory infiltrate in DSS-treated Cx3cr1gfp/gfp and Cx3cr1–/– mice when compared with Cx3cr1+/+ mice (Figure 4B). Since mice lacking CX3CL1 also displayed a reduction in the frequency of both CD11c– and CD11c+ LP macrophages (Supplemental Figure 3) and increased bacterial translocation to the mLN (Supplemental Figure 5A), we investigated the response of these mice to DSS. Consistent with the increased sensitivity of Cx3cr1gfp/gfp and Cx3cr1–/– mice, Cx3cl1–/– mice also displayed significantly enhanced sensitivity to DSS on days 3–5 (Supplemental Figure 5B). These data demonstrate that interfering with the CX3CR1/CX3CL1 axis results in decreased recruitment and/or retention of macrophages to the intestinal LP, implying that there may be cells in the intestine that secrete CX3CL1 and recruit CX3CR1-expressing LP macrophages. To directly probe this possibility, we isolated RNA from intestinal epithelial cells (IECs) or total LP cells (non-IEC fraction) and performed qRT-PCR for Cx3cl1. Interestingly, we observed that both IECs and LP cells expressed robust levels of Cx3cl1 mRNA (Supplemental Figure 5C). Collectively, these data suggest that regulation of CX3CL1 expression by IECs (27) or LP cells or the regulation of CX3CR1 expression by LP macrophages may have significant effects on macrophage homeostasis and intestinal inflammation.
Increased susceptibility to DSS-induced colitis in CX3CR1-deficient mice. (A) Severity of colitis in DSS-treated Cx3cr1gfp/gfp and Cx3cr1–/– mice or their respective Cx3cr1+/+ age- and vendor-matched control mice, as measured by stool consistency, presence of fecal blood, and weight loss. (B) Colon histology of DSS-treated Cx3cr1gfp/gfp, Cx3cr1–/–, and Cx3cr1+/+ mice. Original magnification, ×400. Data are representative of 3 independent experiments. Error bars represent SEM. *P < 0.05.
Enhanced IL-17A–dependent colitis in CX3CR1-deficient mice. Since Th17 cells and Foxp3+ Tregs play important roles in regulating intestinal inflammation (3), we examined these T cell subsets in the steady state and during intestinal inflammation. Interestingly, under steady-state conditions, CX3CR1-deficient mice showed no significant differences in LP CD4+ Th17 cell or Foxp3+ Treg populations (Supplemental Figure 6) or IFN-γ–producing Th1 cells (data not shown). However, upon induction of intestinal inflammation, a 3.4-fold increase in colonic IL-17–producing CD4+ T cells was observed in Cx3cr1gfp/gfp mice, but not Cx3cr1+/+ mice (Figure 5, A and B). Additionally, there was a trend toward reduced colonic Foxp3+ Tregs in Cx3cr1gfp/gfp mice during DSS treatment; however, this did not reach statistical significance (Figure 5, A and B). Since innate lymphoid cells are a recently appreciated source of IL-17 that contribute to the pathogenesis of intestinal inflammation (28), we investigated whether these cells were an additional source of IL-17A in colitic Cx3cr1gfp/gfp mice. As shown in Supplemental Figure 7, CD3–RORγt+ innate lymphoid cells were not an abundant source IL-17A in Cx3cr1gfp/gfp mice; thus, IL-17A is predominantly produced by CD4+ LP T cells in colitic CX3CR1-deficient mice.
IL-17 responses contribute to enhanced colitis in Cx3cr1gfp/gfp mice. (A) Flow cytometry of intracellular Foxp3 or IL-17A by colonic CD4+ T cells from Cx3cr1gfp/gfp or Cx3cr1+/+ mice. Numbers in outlined areas indicate percentage of cells in gate. (B) Percentage of IL-17A+ and FoxP3+ cells in the large intestine of Cx3cr1gfp/gfp and Cx3cr1+/+ mice. (C) Severity of colitis in DSS-treated Cx3cr1gfp/gfp or Cx3cr1+/+ mice, in the presence of neutralizing IL-17A antibody or isotype control. Data are representative of 2 (C) or 3 (A and B) independent experiments. Error bars represent SEM. *P < 0.05 versus respective controls.
To investigate the possibility that the increased disease severity in CX3CR1-deficient mice is related to augmented IL-17A production, we induced acute colitis in Cx3cr1gfp/gfp and Cx3cr1+/+ mice and injected neutralizing antibody against IL-17A or isotype control IgG1 at days 0, 2, and 4. Figure 5C shows that while IgG1 treatment did not affect the severity of the disease in Cx3cr1gfp/gfp and Cx3cr1+/+ mice (when compared with Figure 4A), IL-17A neutralization significantly ameliorated the enhanced colitis symptoms in Cx3cr1gfp/gfp mice, reducing them to levels comparable to those in Cx3cr1+/+ mice. This neutralization also significantly ameliorated colitis symptoms in Cx3cr1–/– mice (Supplemental Figure 8). Importantly, IL-17A neutralization had little to no effect in modulating colitis in Cx3cr1+/+ mice (Figure 5C), thus emphasizing an important and unique role for IL-17A–producing CD4+ T cells in enhanced colitis in CX3CR1-deficient mice. To further confirm that CD4+ T cells were involved in exacerbating colitis in CX3CR1-deficient mice, we treated Cx3cr1gfp/gfp mice with CD4-depleting antibody and monitored disease activity. Complete depletion of CD4+ T cells in Cx3cr1gfp/gfp mice (Supplemental Figure 9A) significantly reduced colonic inflammation as assessed by DAI (Supplemental Figure 9B). Therefore, CD4+ T cells are the major IL-17–producing cells contributing to the pathogenesis of colitis in CX3CR1-deficient mice.
Transfer of Cx3cr1+/+ macrophages suppresses colitis in CX3CR1-deficient mice. In order to probe the relationship between the reduction in LP macrophages and the increased susceptibility to DSS-induced colitis, we transferred Cx3cr1+/+ BMDMs into Cx3cr1gfp/gfp mice 1 day prior to and 1 day after initiating DSS treatment and followed clinical signs of intestinal inflammation. Cx3cr1+/+ BMDMs exhibited a phenotype of CD45hi, CD11bhi, F4/80hi, MHC IIint, CX3CR1+, CD115+, CD135int, CD117–, indicating an intermediate phenotype between bone marrow precursors and tissue macrophages. These cells expressed low levels of CX3CR1 prior to transfer; however, CX3CR1 was upregulated after their differentiation in vivo (data not shown). This expression pattern is consistent with CX3CR1 expression increasing initially as MDPs differentiate into blood monocytes and further as blood monocytes differentiate into intestinal LP macrophages (data not shown).
Beginning at 3 days following DSS administration, Cx3cr1gfp/gfp mice that received transfer of Cx3cr1+/+ BMDMs were significantly protected from colitis when compared with Cx3cr1gfp/gfp mice not receiving transferred macrophages (Figure 6). The protection afforded to Cx3cr1gfp/gfp mice by Cx3cr1+/+ macrophages was similar to that afforded by IL-17A neutralization. In contrast to Cx3cr1+/+ BMDMs, transfer of Cx3cr1gfp/gfp BMDMs did not provide any detectable protection to Cx3cr1gfp/gfp mice, even though Cx3cr1gfp/gfp macrophages were equally viable as Cx3cr1+/+ macrophages and capable of normal TNF production in response to LPS (data not shown). Additionally, transfer of either Cx3cr1gfp/gfp or Cx3cr1+/+ BMDMs into Cx3cr1+/+ mice (Supplemental Figure 10) showed no detectable effect on modulating DSS-induced colitis. Collectively, these data demonstrate that the expression of CX3CR1 on transferred macrophages is required for protecting CX3CR1-deficient mice from increased sensitivity to DSS-induced colitis. Furthermore, homing of transferred macrophages to the intestine may be important for their ability to protect from colitis, since Cx3cr1+/+ BMDMs were able to migrate to the intestine, as demonstrated by recovery of CDllb+CD11c– APCs from the LP at day 5 (Supplemental Figure 11).
LP macrophage deficiency contributes to enhanced colitis in Cx3cr1gfp/gfp mice. Severity of colitis in DSS-treated Cx3cr1gfp/gfp mice after adoptive transfer of Cx3cr1gfp/gfp or Cx3cr1+/+ BMDMs. Data are representative of 2 independent experiments. Error bars represent SEM. *P < 0.05 versus respective controls.