Dynamic Changes in Macrophage Activation and Proliferation during the Development and Resolution of Intestinal Inflammation

Macrophages (Mφs) accumulate at sites of inflammation, and, because they can assume several functionally distinct states of activation, they can either drive or restrain inflammatory responses. Once believed to depend on the recruitment of blood monocytes, it is now clear that the accumulation of Mφs in some tissues can result from the proliferation of resident Mφs in situ. However, little is known about the proliferation and activation state of Mφ subsets in the gut during the development and resolution of intestinal inflammation. We show that inflammatory Mφs accumulate in the large intestine of mice during the local inflammatory response to infection with the gastrointestinal nematode parasite Trichuris muris. Classically activated Mφs predominate initially (as the inflammation develops) and then, following worm expulsion (as the inflammation resolves), both the resident and inflammatory populations of Mφs become alternatively activated. A small but significant increase in the proliferation of inflammatory Mφs is seen but only during the resolution phase of the inflammatory response following both worm expulsion and the peak in Mφ accumulation. In contrast to recent studies in the pleural and peritoneal cavities, the proliferation of resident and alternatively activated Mφs does not increase during the inflammatory response. Furthermore, in CCR2−/− mice, monocyte recruitment to the gut is impeded, and the accumulation of alternatively activated Mφs is greatly reduced. In conclusion, the recruitment of blood monocytes is the principle mechanism of Mφ accumulation in the large intestine. This study provides a novel insight into the phenotype and behavior of intestinal Mφ during infection-driven inflammation.

M acrophages (Mws) are mononuclear phagocytes of the innate immune system and are involved in hostdefense, metabolism, and the homeostatic regulation of healthy tissues. Playing diverse and contrasting roles, Mws can initiate, amplify, and regulate the adaptive immune system and both drive and resolve inflammatory responses. The gut is the largest reservoir of Mws in the body (1), and intestinal Mws play a key role in driving the pathogenesis of inflammatory bowel disease (2).
Mws can assume several functionally different states of activation that are regulated by the prevailing cytokine milieu and other factors that are present at sites of inflammation. Mws respond to IFN-g, with or without LPS, to become classically activated (3,4). Classically activated Mws (M1s) play a vital role in Th1-mediated immunity against intracellular pathogens and are characterized by the expression of inducible NO synthetase (iNOS) (3,4). In contrast, IL-4 and IL-13 induce the alternative activation of Mws by signaling through IL-4Ra (4), the common subunit of their receptors. Associated with both Th2-mediated allergic reactions and responses to a range of phylogenetically distinct helminth parasites (5), alternatively activated Mws (M2s) are characterized by their expression of arginase-1, resistin-like molecule a (RELMa), and Ym-1 (4).
Distinct resident and inflammatory subpopulations of Mws exist in tissues, including the gut. Much of our understanding of the functional specialization of Mw subsets has been through the development of CX3CR1 gfp/+ transgenic mice, which express eGFP under the control of the CX3CR1 promoter (6). CX3CR1 hi resident Mws and CX3CR1 int inflammatory Mws can be easily identified by their differential expression of eGFP (7). Resident Mws in the gut are involved in homeostasis and the prevention of inflammatory reactions against commensal bacteria and food proteins (8). In most tissues (including the brain, liver, spleen, and lungs), resident Mws are derived during embryogenesis from cells in the yolk sac and fetal liver, and after birth, they are maintained by self-renewal (9)(10)(11). However, the origin of gut-resident Mws appears to be unique because they are derived from Ly6C hi CX3CR1 lo blood monocytes (12)(13)(14).
During the development of colitis, inflammatory Mws accumulate in the inflamed mucosa, where they produce TNF-a and other proinflammatory mediators (14)(15)(16)(17). They are recruited from Ly6C hi CCR2 hi CX3CR1 lo blood monocytes in a CCR2-dependent mechanism and drive the inflammatory response (14,15,17). However, during an inflammatory response in the pleural and peritoneal cavities, resident Mws proliferate. Therefore, in these tissues, Mw accumulation during inflammation can be accomplished independent of monocyte recruitment (18)(19)(20). However, in the gut, it remains to be determined whether the proliferation of Mws acts in tandem with the recruitment of blood monocytes to Trichuris muris, a natural nematode parasite of mice that resides in the cecum and proximal colon, is a model for the human whipworm Trichuris trichiura, which infects as many as one billion people worldwide (21). Resistance to a high-level infection with T. muris varies considerably between different strains of mouse. Many strains, such as BALB/c, mount a protective Th2 response to T. muris, leading to the rapid expulsion of the parasite, whereas others, such as C57BL/6, mount a mixed Th1/Th2 response and expel the parasite more slowly. In contrast, susceptible strains, such as AKR, mount an inappropriate Th1 response and fail to expel T. muris (22,23). Furthermore, a low-level infection also induces a Th1 response, and this confers susceptibility to all strains of mouse (24). Importantly, regardless of the underlying adaptive immune response, the large intestine becomes inflamed as Mws, and other leukocytes, accumulate in the tissue (23).
By exploiting this natural model of intestinal inflammation, we describe the dynamic changes that take place to Mw subtypes and their activation states as inflammation develops and resolves. Furthermore, we use CX3CR1 gfp/+ transgenic mice and multiparameter flow cytometry to distinguish among resident, inflammatory, and M2 subsets of Mws and assess their proliferation in the intestine.

Mice
Specific pathogen-free AKR, BALB/c, and C57BL/6 mice were purchased from Harlan. CX3CR1 gfp/+ mice were bred at the University of Manchester. CCR2 2/2 mice were purchased from The Jackson Laboratory. All strains of mouse were maintained in individually ventilated cages. Only the males were used in experiments when they were 6-12 wk old. The mouse studies were reviewed and approved by the Home Office and performed under the strict legal requirements of the Animal (Scientific Procedures) Act 1986 (as amended).

Parasite
The E strain of T. muris was maintained as described previously (25). T. muris excretory/secretory (E/S) Ags were prepared by culturing adult worms in vitro at 37˚C for 4 h (25). T. muris eggs were administered, by oral gavage, resulting in either a low-level infection (35 eggs given) or a high-level infection (200 eggs given).

Cell culture
Mesenteric lymph node (MLN) cells were cultured and stimulated with 50 mg/ml T. muris E/S Ags for 48 h as previously described (23). The culture supernatants were harvested and stored at 220˚C until they were assayed for cytokines.

Multiplex quantification of cytokines
A Cytometric Bead Array kit (BD Biosciences, Oxford, U.K.) was used in accordance with the manufacturer's instructions to assay cytokines using an LSR II flow cytometer (BD Biosciences).

Isolation of lamina propria leukocytes
Lamina propria leukocytes (LPLs) were isolated from the proximal colon and cecum by enzymatic digestion as previously described (14,15).

Proliferation
Two approaches were taken to measure proliferation. Firstly, mice were injected i.p. with BrdU, which is incorporated into the newly synthesized DNA of replicating cells during the S phase of the cell cycle. The mice were killed 4 h later, and an Ab was used to detect the BrdU in the DNA of Mws by flow cytometry (as described next). Secondly, an Ab was used to measure Ki-67 in Mws by flow cytometry. This nuclear protein regulates cell division and is present during all active phases of the cell cycle (G 1 , S, G 2 , and M) but is absent from quiescent cells (G 0 ).

Immunohistochemistry
Immunohistochemistry was performed on frozen cross-sections of proximal colon using standard immunoperoxidase techniques as described previously (23). The following primary Abs were used: biotin rat, anti-mouse CD4 mAb (5 mg/ml; BD Biosciences), biotin rat, anti-mouse CD45 mAb (2 mg/ml; BD Biosciences), biotin rat, anti-mouse F4/80 mAb (2 mg/ml; AbD Serotec, Oxford, U.K.), rabbit, anti-mouse RELMa polyclonal Ab (2 mg/ml; PeproTech), goat, anti-mouse Arginase-1 polyclonal Ab (1 mg/ ml; Santa Cruz Biotechnology, from Insight Biotechnology, Wembley, U.K.), goat, anti-mouse Ym-1 (Chitinase 3-like 3/ECF-L) polyclonal Ab (2 mg/ml; R&D Systems, Abingdon, U.K.), or rabbit, anti-mouse iNOS polyclonal Ab (1 mg/ml; Santa Cruz Biotechnology). The following secondary Abs were then used: for Arginase-1 and Ym-1 staining, we used biotin rabbit, anti-goat IgG F(ab9) 2 (1/2000 v/v; Millipore, Watford, U.K.), and for iNOS and RELMa, we used biotin goat, anti-rabbit IgG F(ab9) 2 (1/600 v/v; Santa Cruz Biotechnology). The appropriate isotype control mAbs or polyclonal control IgGs were used in parallel sections. The color development was monitored and was stopped, by washing in PBS, before any false-positive staining occurred in the isotype control sections. The sections were counterstained in Haematoxylin QS (Vector Laboratories). After randomization and blinding of the slides, the number of positively stained cells was determined in each section by light microscopy. The staining was performed in triplicate, and all of the positively stained cells in each section were counted (as a guide, there are ∼200 crypts in each section).

Statistics
Statistical analysis was performed by the Kruskal-Wallis test with Dunn's posttest (using GraphPad Prism software; GraphPad).

Results
Following infection with T. muris, Mws accumulate in the large intestine of C57BL/6 mice, where they are the predominant type of infiltrating leukocyte The detection of CD45, F4/80, and CD4 by immunohistochemistry allowed the number of leukocytes, Mws, and Th cells, respectively, to be quantified in the proximal colon of C57BL/6 mice. In uninfected mice, .90% of the leukocytes were Mws (Fig. 1). Following a high-level infection with T. muris, leukocytes accumulated in the large intestine. There was a significant increase in the number of both Mws and Th cells in the proximal colon 21 d postinfection, and ∼80% of the leukocytes were Mws (Fig. 1). Similar values were found in a previous study in BALB/c and AKR strains of mouse (23). Eosinophils (analyzed by the immunohistochemical staining of Siglec-F) also accumulated in the large intestine postinfection (in uninfected mice, there were 0.2 6 0.1 Siglec-F + cells per crypt compared with 2.8 6 1.7 cells/crypt 21 d postinfection, data not shown). Eosinophils are known to express F4/80 as well as Mws. However, because Siglec-F + cells were much less abundant than F4/80 + cells, only a small fraction of the F4/80 + cells were potentially eosinophils.
Following a high-level infection, the adaptive immune response and the ability to expel T. muris are strain dependent After day 35 postinfection, Ag-stimulated MLN cells from AKR mice released high levels of IFN-g and IL-17A. Furthermore, on day 42, IL-13, but not IL-5, was also released (Fig. 2), revealing that AKR mice mounted strong Th1 and Th17 responses (and also a delayed and muted Th2 response) to the parasite. In contrast, Ag-stimulated MLN cells from BALB/c mice produced high levels of IL-5 and IL-13, but not IFN-g postinfection. This was accompanied by a small but significant increase in IL-17A on day 42 (Fig. 2). Therefore, BALB/c mice mounted a strong Th2 response (and also a weak and delayed Th17 response) to T. muris. MLN cells from C57BL/6 mice released high levels of all four cytokines after day 21 postinfection (Fig. 2). Therefore, C57BL/6 mice mounted strong Th1, Th2, and Th17 responses. AKR mice failed to expel T. muris and a chronic infection ensued. In contrast, BALB/c and C57BL/6 mice were both resistant. However, BALB/c mice expelled the parasite more rapidly than C57BL/6 mice (Fig. 3A).
The emergence of M1s and M2s in the large intestine postinfection follows a distinct pattern in each strain of mouse, reflecting the kinetics of worm expulsion and/or the underlying adaptive immune response Immunohistochemical staining for the M1 marker iNOS and the M2 markers Arginase-1, Ym1, and RELMa, allowed these cells to be quantified in the proximal colon. In all three strains of mouse, there was a significant increase in the number of iNOS + mononuclear leukocytes (henceforth referred to as M1s). In BALB/c and C57BL/6 mice, the number of M1s reached a peak 21 d postinfection and then subsequently decreased. In contrast, in AKR mice, the M1s emerged later and they persisted (Fig. 3D). In each of the three strains of mouse, there was a trend toward an increase in the number of Arginase-1 + , Ym-1 + , and RELMa + mononuclear leukocytes postinfection. However, in AKR mice, the only significant increase was for Ym-1 + cells (Fig. 3D), and therefore, it is uncertain whether M2s emerged in this strain of mouse. In BALB/c and C57BL/6 mice postinfection, the accumulation of M2s in the large intestine (based on all three M2 markers) was clearer, and it reached a peak following worm expulsion (Fig. 3A, 3D). Surprisingly, Ym1 + cells were the least abundant in the most Th2-biased strain of mouse, namely BALB/c, reinforcing the need to analyze multiple markers to define alternative activation. Interestingly, in C57BL/6 mice, M1s emerged in the gut during worm expulsion, whereas M2s were most abundant following worm expulsion after the number of M1 had diminished. Both before and postinfection, the M1s and M2s were mainly situated in the lamina propria (Fig. 3B) and smooth muscle (not shown) compartments of the gut: they were rarely encountered in the intraepithelial niche of the mucosa (Fig. 3B). After day 21 postinfection, some iNOS + and RELMa + (but not Arginase-1 + or Ym-1 + ) eosinophil-like polymorphonuclear leukocytes were also observed (Fig. 3C). However, these cells were not quantified. LPLs were liberated from the lamina propria and stained with a panel of fluorochrome-labeled Abs. A series of gating steps was performed to exclude cell clusters and doublets, select live leukocytes, and exclude eosinophils and dendritic cells (DCs) from the subsequent analysis. The F4/80 + CD11b + cells were defined as Mws and selected for downstream analysis (Fig. 4A). Paradoxically, although the number of leukocytes in the large intestine increases postinfection with T. muris (Fig. 1), infected gut tissue yields fewer leukocytes from the lamina propria than uninfected tissue. As reported previously (23), the immunopathological disruption to the gut postinfection appears to interfere with the isolation of leukocytes from the lamina propria leading to an artificially low yield. Therefore, because it cannot be determined reliably postinfection, the flow cytometry data were expressed not as total numbers of M2s but instead as the relative percentage of M2s within the total Mw population.
The marker RELMa was chosen for the analysis of M2s by flow cytometry because it exhibited similar staining profiles to Arginase-1 and Ym1, yet it revealed the greatest differences between uninfected and infected mice (Fig. 3D). In all three strains of mouse, ∼10% of Mws from the lamina propria of the large intestine, in its resting state, were alternatively activated. In AKR mice, the relative percentage of M2s decreased gradually postinfection (Fig. 4B, 4C). Conversely, in both C57BL/6 and BALB/c mice, the relative percentage of M2s increased postinfection showing that about one-third of the Mws were alternatively activated. Reaching a peak after worm expulsion, the accumulation of M2s reflected the different kinetics of worm expulsion between these two strains of mouse (Fig. 4B, 4C), recapitulating the earlier observations made by immunohistochemistry (Fig. 3).
A minor fraction of CD103 + DCs also expressed RELMa in all three strains of mouse, and there was a small but significant increase in the relative percentage of these RELMa + DCs 42 d postinfection in C57BL/6 and BALB/c mice (Supplemental Fig.  1A-C). Approximately 5% of eosinophils also expressed RELMa, but there was no significant change postinfection (Supplemental Fig. 2).

Alternative activation occurs specifically in the resident and mature inflammatory Mw subpopulations
Using the careful gating strategy described above (Fig. 5A), we went on to investigate which subpopulations of Mws became alternatively activated in response to T. muris infection in CX3CR1 gfp/+ mice (on a C57BL/6 background). The relative percentage of monocytes (P1), immature inflammatory Mws (P2), mature inflammatory Mws (P3), and resident Mws (P4) expressing RELMa was analyzed. Furthermore, to establish whether a Th2 response and worm expulsion was required for the accumulation of M2 in the large intestine, two disparate strategies of T. muris infection were employed: firstly, the familiar high-level infection protocol that resulted in a mixed Th1/Th2 response and worm expulsion [with the same kinetics that was observed for wild-type (WT) C57BL/6 mice (Fig. 3A), not shown]; and secondly, a lowlevel infection protocol that, contrastingly, resulted in a Th1 response and chronic infection (not shown).
Hardly any monocytes (P1) expressed the M2 marker RELMa (Fig. 5D, 5E). In uninfected mice, only a small proportion of Mws (subpopulations P2-P4) were alternatively activated (Fig. 5D, 5E). However, following a high-level infection, M2s emerged, and they were observed in the mature inflammatory (P3) and mature resident (P4) Mw subpopulations (Fig. 5D, 5E). After worm expulsion, approximately half of the Mws within these subpopulations were alternatively activated (Fig. 5E). As late as day 57 postinfection, a significant proportion of the mature inflammatory Mw subpopulation (P3) was alternatively activated (Fig. 5E). M2s also emerged following a low-level (chronic) infection, but this was restricted to the mature resident Mw subpopulation (P4) and was less marked when compared with a high-level (acute) infection (Fig. 5D, 5E). Therefore, the highest level of M2 accumulation was observed following worm expulsion.

A small but significant increase in the proliferation of mature inflammatory Mws occurs in the large intestine following worm expulsion
In uninfected mice, ∼2% of Mws in the large intestine had incorporated BrdU into their DNA. As expected, most of the BrdU + Mws also expressed Ki-67 (Fig. 6A, 6B, Supplemental Fig. 3A). Therefore, a small number of Mws proliferated in the large intestine in its resting state. Postinfection no significant increase in the relative percentage of BrdU + or BrdU + Ki-67 + Mws was detected (Fig. 6A, 6B), suggesting that proliferation does not account for the accumulation of Mws following infection with T. muris in any of the different strains of mouse. Approximately 10 times more Mws were Ki-67 + than BrdU + reflecting the broader scope of Ki-67 as a marker of proliferation than BrdU. At 21 d postinfection, there was a significant increase in the relative percentage of Ki-67 + Mws but only in AKR mice. However, this is difficult to interpret as a bona fide increase in the proliferation of Mws because it was not accompanied by an increase in the number of BrdU + cells (Fig. 6A, 6B).
Despite not observing a significant increase in the percentage of BrdU + Mws postinfection, we investigated whether a small increase could have been overlooked because it was restricted to one of the subpopulations of Mws. Interestingly, after the worms had been expelled, there was a small but significant increase in the percentage of BrdU + Ki-67 + mature inflammatory Mws (P3), suggesting that proliferation may contribute to the accumulation of this subpopulation of Mws in the large intestine postinfection (Fig. 6C, 6D, Supplemental Fig. 3B). However, in contrast to peritoneal and pleural cavity (18,20), the vast majority of BrdU + Mws in the colon were RELMa 2 (Fig. 7), implying that few of the M2s proliferated. Therefore, the accumulation of M2s in the large intestine postinfection is probably not driven by their proliferation in situ.

The accumulation of Mws and M2s in the large intestine postinfection is greatly reduced in CCR2-deficient mice
It has been shown previously that the recruitment of blood monocytes to the intestine is CCR2 dependent (14,15,17). Therefore, we used CCR2 2/2 mice to inhibit monocyte chemotaxis to investigate whether blood-derived monocytes give rise to the Mws and M2s that accumulate in the large intestine postinfection. CCR2 2/2 mice (on a C57BL/6 background) were resistant to a high-level infection with T. muris (not shown) as reported previously (27). Immunohistochemical staining for F4/80 revealed that, in the absence of infection, Mws resided in the lamina propria of the colon in both CCR2 2/2 mice and their WT controls (Fig. 8A, 8B). In WT mice, Mws accumulated in the colon, reaching a peak 21 d postinfection (Fig. 8B). In contrast, there was  no significant accumulation of Mws in CCR2-deficient mice (Fig. 8B). Low numbers of RELMa + M2s were detected in the uninfected gut of both CCR2 2/2 and WT mice by immunohistochemistry (Fig. 8C, 8D). In WT mice, the number of M2s increased postinfection, reaching a peak at day 42 (Fig. 8D). However, in CCR2 2/2 mice, there was no increase in the number of M2s in the colon postinfection (Fig. 8D). The significant accumulation of M2s in the large intestine of WT mice, but not CCR 2/2 mice, was confirmed by flow cytometry (Fig. 8E-G). Therefore, to a large extent, the accumulation of M2s in the intestine postinfection is driven by the CCR2-dependent recruitment of monocytes from the blood.

Discussion
Our basic understanding of Mw physiology has been revolutionized by the recent discovery that tissue-resident Mws can proliferate in situ. In some tissues, this acts not only as a mechanism for the maintenance of resident Mw numbers (9-11) but also enables the accumulation of Mws at sites of inflammation independent of monocyte recruitment from the blood (18)(19)(20). However, whether this translates to all inflamed tissues remains to be determined. This study describes the activation state and proliferation of res-ident and inflammatory Mws in the large intestine during both acute and chronic inflammation driven by the nematode parasite T. muris.
In resistant strains of mouse, the expulsion of T. muris precedes the accumulation of M2s, and the peak accumulation of M2s is reached after worm expulsion. In BALB/c mice, the gradual reduction in the number of M2s following worm expulsion probably reflects the return of the gut to a steady state after the loss of the parasites. M2s have been shown to play pivotal role in the expulsion of the gastrointestinal nematode Heligomosoides polygyrus bakeri during Th2 memory responses to a secondary infection (28). However, a role for M2s in the expulsion of the nematode Nippostrongylus brasiliensis is controversial (29,30). In a previous study, we showed that disrupting the function of M2s (by inhibiting their arginase-1 activity) has no effect on the expulsion of T. muris (31). Accordingly, in this study we show that mice are resistant to T. muris even when the accumulation of M2s to the large intestine is inhibited. Therefore, M2s are not required for the expulsion of T. muris. Instead, because of the kinetics of M2 accumulation, our data support a role for M2s in the gut following worm expulsion, during the resolution phase of the inflammatory response. This is consistent with the ability of M2s to restrain the potentially damaging immunopathology following infection with nematode parasites (29,32,33) and a role for M2s in tissue repair and remodeling (34,35).
Interestingly, in C57BL/6 mice, the wavelike accumulation of M1s is observed in the gut, reaching a peak around the time of worm expulsion and then receding as M2s begin to accumulate (during worm expulsion) and then predominate (after worm expulsion). Indeed, a similar transition from M1s to M2s has been observed following infections with parasites as diverse as Taenia crassiceps, Schistosoma mansoni, and Trypanosoma congolense (36,37), and it is possible that the dynamics of M1 and M2 accumulation reflects sequential changes in the local cytokine milieu. However, the factors that drive this switch remain to be determined. It is possible to reprogram polarized Mws in vitro, so that M1s can be transformed into M2s and vice versa, by switching the cytokine stimulus (38). This remarkable plasticity of Mws may also occur in vivo because M2s seem to convert to M1s in artherosclerotic lesions (38). However, it still remains unclear whether the switch from M1 to M2 represents the recruitment of naive Mw precursors or involves the re-education of the same Mws in situ.
Using published approaches to define monocyte and Mw subsets by multiparameter flow cytometry (14,16,26), we demonstrate, for the first time to our knowledge, the dynamic changes that occur to resident and inflammatory gastrointestinal Mws during an inflammatory response to infection. We confirm that CX3CR1 high resident Mws are the predominant population in the uninfected large intestine, although CX3CR1 int inflammatory Mws are also encountered (14,15). Postinfection with T. muris, inflammatory Mws accumulate in the large intestine and become more prevalent than resident Mws. Importantly, we show for the first time, to our knowledge, that both inflammatory and resident Mws become alternatively activated following infection with a gastrointestinal nematode. Furthermore, both inflammatory and resident Mws remain alternatively activated for several weeks after the immunopathology appears to have subsided. That inflammatory Mws can be alternatively activated reveals an interesting and far-reaching paradox because inflammatory Mws are thought to amplify inflammation, whereas M2s are implicated in the resolution of inflammation (7,14,15,17,39).
The proliferation of resident Mws drives the accumulation of Mws in the pleural and peritoneal cavities following infection with filarial nematode parasites (18,19), and the replenishment of Mws in atherosclerotic lesions depends predominantly on local Mw proliferation (40). Therefore, in these models of inflammation, Mw proliferation, rather than monocyte influx, is the principle mechanism underlying the accumulation of Mws. Although the accumulation of Mws during the development of colitis has been shown to involve the recruitment of Ly6C high CX3CR1 low inflammatory monocytes (7,15,17), the potential of local resident Mw proliferation to contribute toward this process has not been investigated previously. During T. muris infection, the accumulation of Mws in the large intestine does not coincide with an increase in the proliferation of resident Mws. Furthermore, the inhibition of monocyte recruitment greatly impedes the accumulation of Mws in the gut. Therefore, monocyte recruitment is the principle mechanism of Mw accumulation during the development of the inflammatory response to T. muris. Nevertheless, following worm expulsion, there is a small but significant increase in the proliferation of inflammatory Mws. Given that, in the large intestine, resident Mws are derived from inflammatory Mws (14), the increase in the proliferation of inflammatory Mws could boost resident Mw numbers in the late stages of inflammation following worm expulsion.
Importantly, we reveal that the vast majority of M2s do not proliferate in the large intestine at any stage either before or postinfection with T. muris. Furthermore, we show that the accumulation of M2s in the large intestine is greatly reduced by disrupting monocyte recruitment to the gut. This is consistent with previous work showing a CCR2-dependent mechanism underlying the recruitment of Ly6C hi CCR2 hi CX3CR1 lo blood monocytes to the colon during an inflammatory response (14,15,17). Therefore,  In mice lacking CCR2, the accumulation of Mws and M2s in the colon postinfection is greatly reduced. CCR2 2/2 and WT control mice (C57BL/6) were either left uninfected or infected with a high level of T. muris ova. Immunohistochemical staining of Mws (F4/80 + cells) was conducted on sections of the proximal colon. Representative photographs of the F4/80 staining are shown in (A), and the quantitative analysis is shown in (B). Immunohistochemical staining of M2s (RELMa + cells) was also performed on sections of the proximal colon. Representative photographs of the RELMa staining are shown in (C), and the quantitative analysis is shown in (D). Scale bars, 100 mm. Cells were isolated from the lamina propria of the cecum and proximal colon, stained with a panel of fluorochrome-labeled Abs, and then analyzed by flow cytometry. Live Mws were analyzed by gating on viability stain-negative CD45 + CD11b + F4/80 + CD103 2 Ly6G 2 Siglec-F 2 cells (as shown in E). Representative plots of RELMa staining are shown in (F), and the data are shown graphically in (G). The values are the means 6 SEM of five mice in each group. *p , 0.05 (CCR2 2/2 compared with WT at the same time point).
mansoni. Taken together, it is becoming clear that the mechanisms that underlie the accumulation of M2s following infection with parasitic nematodes are either tissue specific or parasite species specific.
In summary, this study reveals the dynamic changes that take place to the phenotype of Mw subsets during the initiation, amplification, and resolution of intestinal inflammation. We describe the emergence of M1s during worm infection and M2s following worm expulsion. However, in contrast to previous studies (18,19), in the large intestine, the accumulation of M2s is chiefly dependent on the recruitment of blood monocytes rather than their proliferation. Understanding the mechanisms that control M1/M2 balance will bring the pharmacological manipulation of Mws a step closer. The promotion of antiinflammatory and the restraint of proinflammatory subsets of Mws have exciting potential for the treatment of a range of debilitating inflammatory diseases.