Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2018 Sep 1.
Published in final edited form as: Arthritis Rheumatol. 2017 Aug 1;69(9):1762–1771. doi: 10.1002/art.40151

Increased F4/80hi macrophages is associated with suppression of serum transfer induced arthritis in mice with Flip reduced in myeloid cells

Qi-Quan Huang 1, Robert Birket 1, Renee E Doyle 1, G Kenneth Haines 2, Harris Perlman 1, Bo Shi 1, Philip Homan 1, Lianping Xing 3, Richard M Pope 1
PMCID: PMC5575965  NIHMSID: NIHMS875710  PMID: 28511285

Abstract

Objective

Macrophages are critical in the pathogenesis of rheumatoid arthritis (RA). We recently demonstrated that FLIP (FLICE-like inhibitory protein) is necessary for the differentiation and/or survival of macrophages. We also identified that FLIP is highly expressed in RA synovial macrophages. This study was performed to determine if the reduction of Flip in macrophages would reduce synovial tissue macrophages and ameliorate serum transfer induced arthritis (STIA).

Methods

Mice with Flip deleted in myeloid cells (Flipf/fLysMc/+ mice) and littermate controls were employed. STIA was induced by intraperitoneal injection of K/BxN serum. Arthritis was evaluated by clinical score and change in thickness of the ankles. Joints were examined by histology and immunohistochemistry. Cells were isolated from the ankles and bone marrow and examined by flow cytometry, qRT-PCR or Western blot.

Results

In contrast to expectations, Flipf/fLysMc/+ mice developed more severe arthritis early in the clinical course, however the peak arthritis was attenuated and the resolution phase more complete. Prior to the induction of STIA, tissue resident macrophages were reduced. Further at day 9 post arthritis induction the number of F4/80hi macrophages in the joints of the Flipf/fLysMc/+ mice was not decreased, but increased. Flip was reduced in the F4/80hi macrophages in the ankles of the Flipf/fLysMc/+ mice, while F4/80hi population expressed an anti-inflammatory phenotype in both the Flipf/fLysMc/+ and control mice.

Conclusions

These observations suggest that reduction of FLIP in macrophages, by increasing the number of anti-inflammatory macrophages, may be an effective therapeutic approach to suppress inflammation, depending upon the disease stage.

INTRODUCTION

Macrophages are critical to the pathogenesis of rheumatoid arthritis (RA). Macrophage numbers are increased in the synovial tissues of patients with RA (1, 2) and increased numbers of synovial tissue macrophages predict future joint damage (3). Further, the reduction of subsynovial lining macrophages observed after the initiation of therapy in patients with RA correlated with clinical benefit, while there was no correlation with other cell types such as B or T lymphocytes (4). RA synovial tissue macrophages express increased levels of pro-inflammatory chemokines and cytokines such as TNFα and IL-6, and neutralization of these cytokines is effective therapy in patients with RA (1, 2). Even though the local environment of the RA joint appears pro-apoptotic because of increased reactive oxygen and nitrogen species, RA synovial macrophages appear resistant to apoptotic cell death (5, 6). Previously we demonstrated that RA synovial macrophages express both Fas and FasL (6). They also highly express FLIP (FLICE-like inhibitory protein), which is critical for protecting RA synovial macrophages from Fas mediated apoptosis (6). Together these observations suggest that regulating the level of FLIP in macrophages might be an effective therapeutic approach to reduce the number of macrophages in patients with RA and other forms of inflammatory arthritis. Of relevance to this possibility, Flip (CFLAR) has recently been identified as an RA risk allele (7).

To examine the in vivo role of FLIP in macrophages, we generated genomically modified mice with the Flip gene floxed (8). Mice with one allele of Flip deleted and the other floxed (Flipf/d) were crossed with LysMcre (LysMc/+) mice to ensure the optimal depletion of the floxed allele in myeloid cell lineage (8). The Flipf/dLysMc/+ mice developed severe postnatal growth retardation and premature death, leukocytosis with multi-organ neutrophil infiltration, severe reduction of macrophages in peripheral organs together with increased extramedullary myelopoiesis (8). We were unable to detect Flip deficient macrophages and bone marrow progenitor cells failed to differentiate into macrophages (8) in vitro when Flip was deleted. These observations demonstrate that Flip is necessary for macrophage differentiation and survival (8).

Since the Flipf/dLysMc/+ mice were severely ill, we generated Flipf/fLysMc/+ mice which resulted in a less extreme phenotype. Flipf/fLysMc/+ mice were employed to generate serum transfer induced arthritis (STIA) employing anti-glucose 6 phosphate isomerase (anti-GPI) positive serum from K/BxN mice. The onset of arthritis was more rapid in the Flipf/fLysMc/+ mice and the number of synovial tissue neutrophils was increased early in the clinical course, compared with littermate controls. However, peak arthritis was reduced in the Flipf/fLysMc/+ mice, as was the arthritis during the resolution phase. In contrast to expectations, this was not due to a reduction of macrophages. In fact the number of F4/80hi macrophages in the joints of the Flipf/fLysMc/+ mice with arthritis were increased. Further, Flip was decreased in the F4/80hi macrophages in the ankles of the Flipf/fLysMc/+ mice, however, the F4/80hi population exhibited an anti-inflammatory phenotype in both groups of mice. These observations suggest that the expression of FLIP, attenuates the development of F4/80hi macrophages under inflammatory conditions and that an increase of F4/80hi macrophages in the Flipf/fLysMc/+ mice results in amelioration of arthritis in the STIA model.

MATERIALS AND METHODS

Mice with Flip conditional knockout in myeloid lineage

We generated a genetically modified mouse line with Flip floxed (Flipf/f) on a C57Bl/6 background as described previously (8). Mice deficient in FLIP in myeloid cells were generated by crossing Flipflox/flox (Flipf/f) mice with the LysMc/+ mice (9). Littermates or gender and age matched mice with Flipflox/+LysMc/+ or Flipf/fLysM+ served as controls. All genotyping was performed by PCR employing genomic DNA extracted from tail biopsies, as described (8). All recombinant DNA and animal procedures were approved by the Office of Research Safety and the Institutional Animal Care and Use Committee of Northwestern University.

K/BxN serum transfer arthritis and analysis

K/BxN mice were generated and anti-GPI serum collected at 8–9 weeks of age as described (10). Arthritis was induced in 7–16 week old Flipf/fLysMc/+ or littermate control mice employing 150 µl of anti-GPI serum administrated intraperitoneally on day 0. The development of arthritis was assessed by measuring hind ankle swelling (thickness, mm) using a caliper and grading the clinical index of all 4 paws/ankles on a scale of 0–3 (maximum = 12) according to previous publications (11, 12). Arthritis was evaluated between days 0 to 14 post-induction. Synovial macrophages were isolated from the ankles before or at day 9 of anti-GPI serum induced arthritis. The expression of IL-10, Resistin-like molecule alpha (Relm-a), Arginase (Arg-1), inducible nitric oxide synthases (iNos) and Flip was determined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Cytokines in ankles were determined by ELISA (DuoSets, R&D, Minneapolis, MN) following manufacturer’s instructions. To do this, ankles were homogenized in PBS containing 1× of a proteinase inhibitor cocktail and supernatants collected by centrifugation as described (11, 13, 14).

Immuno-phenotyping and blood count

Single cell suspensions were prepared from dissected tibias and paws, incubated in 1 mg/ml collagenase in HBSS buffer for 1 hour as described (11, 14, 15). Cell types were determined by immuno-phenotyping and flow cytometric analysis, employing multi-color fluorochrome-conjugated antibodies to CD11b, F4/80, CD64, Ly6C, Ly6G and MHCII (ebioscience or BD Pharmingen). Data were acquired employing BD LSR II flow cytometer and analyzed by FlowJo (TreeStar, Inc.). Synovial macrophage apoptosis was assessed by Annexin V and cell survival by the exclusion of 7-amino-actinomycin (7AAD, BD Pharmingen). The apoptotic cells were identified as Annexin V+ and 7AAD− and the necrotic cells are 7AAD+ examined by flow cytometry (14). Single cell populations were sorted by BD ARIA for genotyping and Western blot analysis. The complete blood counts and differentials were determined by Hemavet 950 from Drew Scientific Inc.

Immunoblot analysis

Immunoblot analysis was performed according to an established protocol (8). The expression of FLIP were recognized by incubation overnight at 4°C with rabbit monoclonal antibody to mouse FLIP (Cell Signaling Technology), followed by anti-rabbit secondary antibodies conjugated with horseradish peroxidase (GE healthcare). After stripping, the same membrane were blotted with antibody to GAPDH (Sigma-Aldrich) for loading adjustment. The specific proteins were detected employing the Enhanced Chemiluminescent Detection Reagent (Amersham Pharmacia).

In vitro differentiation of macrophages from bone marrow cells

In vitro bone marrow differentiated macrophages were generated as described (14). Nucleated bone marrow cells were isolated from femurs and tibias of Flipf/fLysMc/+ or littermate control mice. Bone marrow cells were cultured in RPMI 1640 medium supplemented with 10% (v/v) FBS, 100U penicillin-streptomycin, 0.1% (v/v) of 2-mercaptoethanol, 10 µg/ml polymyxin B and the recombinant mouse M-CSF (20ng/ml, R&D Systems). All bone marrow cells were cultured in duplicate with or without the addition of recombinant mouse TNFα (20ng/ml, R&D Systems) and harvested after 7 days culture at 37°C in a 5% CO2 incubator.

Histological analysis

Ankles were harvested at indicated time points for histological analysis. Ankles were fixed in 10% neutral buffered formalin and then incubated in 10% EDTA decalcification buffer for 2 weeks, embedded in paraffin, and then 4 µm sections were stained with hematoxylin and eosin (H&E). H&E ankle sections was scored 0–5 for joint and extra-articular inflammation, granulocyte infiltration, pannus formation, bone erosion and cartilage destruction as previously described (11, 13, 14). Macrophages in ankles were identified by immunohistochemistry staining employing anti-F4/80 or isotype matched control IgG antibodies. The F4/80+ mononuclear cells were the average of 3 views of the synovium in a 1000× field for each of the ankle slide. The osteoclasts were identified by the Tartrate-resistant acid phosphatase (TRAP) staining, and the frequency of osteoclasts were scored (0–4)(16). All H&E, immunohistochemistry staining and TRAP staining slides were evaluated by blinded investigators.

Data analysis

All quantitative data are presented as mean ± SEM. Statistical analysis between groups was done with 2-tailed Student’s t test, or with one-way ANOVA and Tukey post-test for comparison among multiple groups. Samples that failed the normality test were analyzed by the Mann-Whitney rank sum test. Correlations were determined by Pearson’s linear correlation. Significance levels were set at 0.05.

RESULTS

Flip conditional knock out in myeloid lineage

Since the Flipf/dLysMc/+ mice experienced severe failure to thrive with multi-organ neutrophilic infiltrates (8), Flipf/fLysMc/+ mice were employed to determine the effect of Flip deletion in myeloid cells on the effector phase of inflammatory arthritis employing the serum transfer induced arthritis (STIA). In circulating cells of the Flipf/fLysMc/+ mice, the deleted floxed allele was seen in monocytes but not T or B lymphocytes (Supplemental Figure 1A). In granulocytes the Flip alleles were nearly completely deleted in the Flipf/fLysMc/+ mice, which contrasts with the complete deletion in the Flipf/dLysMc/+ mice (Supplemental Figure 1B). Although mild weight reduction (10~15%), shortened survival, and increased peripheral monocytes and granulocytes were noted in Flipf/fLysMc/+ mice, these features were all less severe than observed in Flipf/dLysMc/+ mice (Supplemental Figure 1C–E and (8)), and spontaneous arthritis was not observed.

Following the injection of anti-GPI serum, modestly increased joint swelling (p < 0.001) at days 2 and 4 post injection was observed in the Flipf/fLysMc/+ mice, compared with the littermate controls, although the clinical score was insufficiently sensitive to detect this difference (Figure 1A). However the peak arthritis scores were less in the Flipf/fLysMc/+ mice, with significantly reduced arthritis during the resolution phase, from day 7 through 14 (Figure 1A). To understand the mechanisms responsible for these differences, ankles were harvested over the course of STIA. Histologically, the inflammation was more severe on day 4, but reduced on day 9 (Figure 1B, C) both in and around the ankle joints of the Flipf/fLysMc/+ mice. Similar differences were observed for neutrophil infiltration, pannus formation and bone erosion (Figure 1C and Supplemental Figure 2). Both IL-1β and IL-6 were significantly increased, prior to day 9 in Flipf/fLysMc/+ mice, compared with littermate controls, although there was no difference of any cytokine on day 9 (Figure 1D). In contrast, IL-10 was significantly reduced on day 4, the peak of arthritis in the Flipf/fLysMc/+ mice, but it was no different from the controls on days 9 (Figure 1D).

Figure 1. Flipf/fLysMc/+ KO mice exhibit enhanced early but attenuated late phase of STIA.

Figure 1

Open in a new tab

(A). The course of the arthritis was documented as the clinical activity score (left panel) and the ankle swelling (Δ ankle thickness in mm for hind ankles, right panel). Data were combined from 5 independent experiments, employing ≥ 40 Flipf/fLysMc/+ or control mice. After sacrificing to harvest samples at days 2 and 4, 18–19 remaining mice were scored on day 9, and 7 mice on day14 post arthritis induction. (B). Representative H&E histology imaging from the ankles of control and Flipf/fLysMc/+ mice harvested at day 4 and day 9. (C). The histological score of ankle joints for joint and extra-articular inflammation, PMN infiltration, pannus formation and bone erosion over the course of STIA (n= 7–16 ankles/group). (D). IL-1β, IL-6 and IL-10 in ankles determined by ELISA. Data are presented as the mean ±1 SE. Significance between the groups was determined by unpaired 2-sided t-test. * represents p< 0.05, ** p < 0.01 and *** p< 0.001 between the indicated groups.

Since bone erosion was reduced at days 9 and 14, and since osteoclasts are derived from myeloid precursors within the inflamed joints, osteoclast numbers were examined. On day 9, a marked decrease (p < 0.01) of TRAP positive osteoclasts was identified in the joints of the Flipf/fLysMc/+ mice compared with those of the littermate controls (Figure 2A). RANKL levels were significantly decreased (p < 0.01) while osteoprotegerin (OPG) levels were increased (p < 0.05) in Flipf/fLysMc/+ mouse ankles (Figure 2B). These observations suggest that the more rapid reduction of inflammation following STIA in Flipf/fLysMc/+ mice was associated with reduced bone erosion due to decreased osteoclastogenesis, which was accompanied by decreased RANKL and increased OPG. These observations may be the result of the reduced inflammation and/or reduced osteoclast survival or differentiation due to Flip depletion.

Figure 2. Attenuated STIA is associated with decreased osteoclastogenesis.

Figure 2

Open in a new tab

(A). Representative TRAP-stained ankle sections from mice that were sacrificed on day 9 post arthritis induction. Numerous TRAP+ osteoclasts (indicated by the arrows) were detected in the ankle joints of control mice (left panel). The frequency of osteoclasts were scored (0–4) and analyzed (right panel, n=7 Flipf/fLysMc/+ mice and 8 controls). (B). The expression levels of RANKL and OPG in ankles at day 9 post arthritis induction mice was quantified by ELISA (n=17 Flipf/fLysMc/+ and 20 control ankles). Data are presented as the mean ±1 SE. Significance was analyzed by unpaired 2-sided t-test for score and OPG analysis, and by Mann-Whitney test for RANKL. *represents p < 0.05 and ** p < 0.01.

Since FLIP is not expressed in neutrophils (Supplemental Figure 3) and Flip deletion in myeloid cells did not diminish neutrophil survival (8) consistent with other reports (17), the effect of myeloid Flip deletion on macrophages in the inflamed joints was examined. We hypothesized that deletion of Flip in myeloid cells would result in reduced synovial tissue macrophages due to increased apoptosis, resulting in diminished arthritis. First we examined joints prior to the onset of arthritis. To our surprise the number of joint macrophages (CD64+CD11b+F4/80+) was not reduced in Flipf/fLysMc/+ mice under homeostatic conditions (Figure 3). Macrophages differentiated from recently recruited circulating monocytes are CD64+CD11b+F4/80+Ly6C+ (18). Synovial tissue resident macrophages under homeostatic conditions (CD64+CD11b+F4/80+Ly6C−MHCII−) are long lived, irradiation resistant and suppress arthritis (19). Although the total number of macrophages was not reduced, the percent of tissue resident macrophages was reduced in Flipf/fLysMc/+ mice prior to arthritis induction (Figure 3).

Figure 3. Flipf/fLysMc/+ ankles exhibit reduced synovial tissue resident macrophages.

Figure 3

Open in a new tab

(A). A representative flow cytometric analysis of ankle macrophages under steady state conditions presenting the gating strategy. After exclusion of doublets and debris, and gating out granulocytes, dendritic cells and B cells, synovial macrophages were identified as CD64+CD11b+F4/80+. After removing Ly6C+ and MHCII+ cells, synovial tissue resident macrophages are identified as CD64+CD11b+F4/80+Ly6C−MHCII−. (B). Analysis of the total synovial macrophages and the percent of synovial tissue resident macrophages from ankles of Flipf/fLysMc/+ mice and the control littermates prior to induction of STIA. (n =13 for Flipf/fLysMc/+ mice and 15 for the controls). Data are presented as the mean ±1 SE. Significance was analyzed by unpaired 2-sided t-test. * represents p < 0.05, ** p < 0.01 and *** p < 0.001 between the indicated groups

Next, the presence of macrophages within the inflamed joints at day 9 was examined by immunohistochemistry. To our surprise, F4/80+ macrophages were actually increased (p < 0.01) at day 9 in the synovial tissue of ankles from Flipf/fLysMc/+ mice compared with the littermate controls (Figure 4A). To better characterize the role of macrophages in STIA in the Flipf/fLysMc/+ mice, the phenotype of macrophages in the joints at 9 days post arthritis induction was examined by flow cytometry. The number of CD11b+F4/80hi macrophages was increased (p < 0.05) in the Flipf/fLysMc/+ mice, while there was no difference in the CD11b+F4/80lo macrophages (Figure 4B). The number of F4/80hi macrophages, as well as the level of expression of F4/80, inversely correlated (p = 0.005–0.001) with joint swelling on clinical exam (Figure 4C).

Figure 4. Flipf/fLysMc/+ mice exhibit increased synovial F4/80+ macrophages by immunohistochemistry and increased F4/80hi synovial macrophages in Flipf/fLysMc/+ mice which correlate with reduced STIA.

Figure 4

Open in a new tab

(A). Immunohistochemistry employing anti-F4/80 was performed to identify the distribution of macrophages in the ankles of mice day 9 post arthritis induction. The left panels are representative anti-F4/80-staining of ankles from control and Flipf/fLysMc/+ mice. The F4/80+ cells are brown. Synovial tissue F4/80+ cells were counted and presented as the average from 3 fields for each ankle (n = 7 Flipf/fLysMc/+ and 8 controls) present at right. (B). Representative flow cytometry images of ankles from control and Flipf/fLysMc/+ mice 9 days post arthritis induction. After gating out granulocytes and CD11b− cells, macrophages were identified as CD64+CD11b+, and further as F4/80hi and F4/80lo. (C). The correlations between arthritis severity (Δ ankle thickness, mm) and the number of F480hi macrophages and the mean florescence intensity (MFI) of F4/80 expression are presented (n = 7 control and 8 Flipf/fLysMc/+ mice). Significance was determined by unpaired 2-sided t-test (A,B), * represents p < 0.05 and ** p < 0.01 between the indicated groups. Pearson linear correlation was performed to determine correlations (C).

In order to determine if there were functional differences between CD11b+F4/80hi and CD11b+F4/80lo macrophages, these populations were isolated by flow cytometry from the ankles 9 days after arthritis induction (Figure 5). In the control mice, by qRT-PCR, the expression of IL-10 and Relm-α were increased (p < 0.001–0.01) in the F/4/80hi population while the expression of iNos was increased (p < 0.01) in those that were F4/80lo (Figure 5A, white bars). Arg-1 was not different between the populations. Similar differences were noted between the F4/80hi and F4/80lo populations in the Flipf/fLysMc/+ mice (Figure 5A, dark bars). In prior studies (8), under homeostatic conditions, any macrophages isolated from Flipf/dLysMc/+ mice still expressed Flip. To our surprise Flip, although present, was significantly (p < 0.05) reduced in the F4/80hi macrophages from the Flipf/fLysMc/+ mice compared with the littermate controls (Figure 5B). No significant difference of Flip was observed in the F4/80lo macrophages between the Flipf/fLysMc/+ and control mice (Figure 5B). To understand where the change in the expression of Flip occurred, monocytes from Flipf/fLysMc/+ and control mice were isolated from bone marrow, which are the immediate precursors of circulating monocytes, by flow cytometry. Compared with blood T lymphocytes, FLIP was variably expressed in the bone marrow derived monocytes from both Flipf/fLysMc/+ and control mice (Supplemental Figure 3). Since the monocyte derived macrophages observed in STIA derive primarily from circulating monocytes which express FLIP in both the Flipf/fLysMc/+ and control mice, it is likely that the reduction of Flip in Flipf/fLysMc/+ F4/80hi macrophages occurs following ingress to the inflammatory environment. Since circulating monocytes demonstrate a deletion of one Flip allele, we speculate that after arriving in the inflamed joints that increased expression of Cre driven by the LysM promoter may result in deletion of the second allele.

Figure 5. During STIA, F4/80hi synovial macrophages exhibit an M2-like phenotype and reduced Flip expression.

Figure 5

Open in a new tab

Macrophages were purified by flow cytometry from the ankles of control and Flipf/fLysMc/+ mice 9 days post STIA induction. Macrophages were gated as CD11b+, CD64+ F4/80hi and F4/80lo as in Figure 4B. (A). The expression of IL-10, Resistin-like molecule alpha (Relm-a), Arginase (Arg-1) and inducible nitric oxide synthases (iNos) determined by qRT-PCR, presented as the fold of expression normalized to the F4/80hi cells of the control mouse group. Data were compared between the F4/80hi and F4/80lo populations of the control or Flipf/fLysMc/+ mice for each molecule (n=6 control and 3 Flipf/fLysMc/+ mice). Significance was determined by unpaired 2-sided t-test (B). The expression of Flip in the same samples was also determined by qRT-PCR, presented as fold of expression normalized to control F4/80hi cells. Analysis was performed by one-way ANOVA, and Tukey post-test. * represents p< 0.05, ** p < 0.01 and *** p< 0.001 between the indicated groups

Ankles were examined to determine if reduction of Flip resulted in increased macrophage apoptosis during STIA. There was no increased apoptosis or necrosis of the macrophages in the ankles of the Flipf/fLysMc/+ mice compared with the controls at day 9 of STIA, consistent with the ability of the F4/80hi macrophages in the Flipf/fLysMc/+ mice to survive despite the reduction of Flip (Figure 6A). Next we used bone marrow derived cells from control or Flipf/fLysMc/+ mice which were in vitro differentiated into macrophages in the presence of M-CSF, with or without TNFα, to simulate inflammatory conditions. After 7 days, as expected in the presence of reduced Flip, the number of macrophages from the Flipf/fLysMc/+ were decreased (Figure 6B). However, in contrast to culture in the absence of TNFα, macrophages expressing reduced levels of FLIP survived (Figure 6B). These observations suggest that in absence of inflammation macrophages expressing FLIP preferentially survived and expanded, while under inflammatory conditions, macrophages with reduced FLIP are capable of survival.

Figure 6. No difference in the apoptosis or necrosis of synovial macrophages at day 9 of STIA between Flipf/fLysMc/+ and control mice.

Figure 6

Open in a new tab

(A). Apoptotic cells were defined as Annexin V+ and 7AAD− and necrotic cells as Annexin V+ and 7AAD+ in CD64+CD11b+F4/80hi and F4/80lo synovial macrophages, harvested at 9 days post arthritis induction. Representative flow images for apoptosis defined in the F4/80hi population of control and Flipf/fLysMc/+ mice are presented in the panels on the left and the percent of apoptotic or necrotic cells in the panels on the right. This analysis was from 5 control and 7 Flipf/fLysMc/+ mice. Significance was determined by unpaired 2-sided t-test. (B). Bone marrow cells from Flipf/fLysMc/+ or littermate control mice differentiated for 7 days in the presence of M-CSF, with or without TNFα. FLIP expression were determined by Western blot. The blot presented is representative of 2 independent experiments.

DISCUSSION

Since FLIP is highly expressed in RA synovial tissue macrophages, protecting them against Fas-FasL mediated apoptosis (5, 6), we reasoned that suppression of FLIP in macrophages within the inflamed joint would be therapeutically beneficial by reducing the number of macrophages. The effective deletion of Flip in myeloid cells (Flipf/dLysMc/+ mice) resulted in a marked reduction of macrophages and a dramatic phenotype, including early mortality due to multi-organ neutrophil infiltrates (8). Therefore we employed Flipf/fLysMc/+ mice which exhibited less severe phenotypic changes. To our surprise, the onset of arthritis in the Flipf/fLysMc/+ mice was more abrupt, while peak arthritis was reduced and the resolution phase was more complete. Unexpectedly under homeostatic conditions, before the induction of STIA, there was no reduction of total synovial tissue macrophages in the Flipf/fLysMc/+ mice, although tissue resident, arthritis-suppressive (19), macrophages were reduced. Further, during the resolution phase of the arthritis an increase of F4/80hi macrophages was found in the joints of the Flipf/fLysMc/+ mice which inversely correlated with disease activity. This population exhibited a reduction of Flip and an M2-like phenotype, which may have been responsible for the enhanced resolution of STIA in these mice. However, M1/M2 polarization of bone marrow differentiated macrophages did not affect Flip (data not shown). Since IL-10 was not increased at day 9 in the joints of the Flipf/fLysMc/+ mice, it is possible that the change from day 4 was important although it is likely that other mechanisms are also responsible. These observations cannot be generalized since other forms of arthritis were not examined.

Recent studies have demonstrated in many tissues that under homeostatic conditions tissue resident macrophages are F4/80hi (18, 20–22). Microglia are MHCII− while those in other tissues express MHCII at variable levels (18). By fate mapping, there is agreement that microglia originate from the yolk sac, and that those in the intestine are derived from fetal hematopoietic stem cells, although there is controversy as to the origin of other tissue resident macrophages including those in the lung, liver and spleen (18, 21). All agree that tissue resident macrophages are long lived, self-populating and radiation resistant. While fate mapping has not been employed for synovial tissue, under homeostatic conditions, CD11b+CD64+Ly6C−MHCII− synovial tissue macrophages are long lived and radiation resistant, suggesting that they are indeed tissue resident macrophages (19). Under homeostatic conditions, a portion of these cells are F4/80hi (Figure 3A) in control and Flipf/fLysMc/+ mice. It remains to be determined, under conditions in which inflammation resolves, to what degree the F4/80hi macrophages derived from circulating monocytes take on the characteristics and function of bona fide tissue resident macrophages.

A previous study of STIA demonstrated that Ly6C− monocytes were necessary and sufficient for the initiation and propagation of the disease, although neutrophils and Ly6C+ monocytes contributed to disease pathogenesis (19). In the Flipf/fLysMc/+ mice, under homeostatic conditions, Ly6C+ classical monocytes are increased. Non-classical, Ly6C− monocytes are critical for the initiation of STIA, and CCR2− mice develop STIA comparably to the wild type controls (19). Therefore, it seems unlikely that the increase of Ly6C+ monocytes, which are also CCR2+, observed in the Flipf/fLysMc/+ mice prior to arthritis initiation (Supplemental Figure 1), contributed to the increased arthritis observed on days 2 and 4. It is more likely that the more severe arthritis observed early in the clinical course of the Flipf/fLysMc/+ mice was due to the reduction of the tissue resident macrophages, since the deletion of MHCII− tissue resident macrophages prior to disease onset resulted in increased STIA (19). Additionally the Flipf/fLysMc/+ mice exhibit increased circulating neutrophils which may also have contributed to the increased arthritis early in the clinical course.

During active inflammation in STIA, monocytes recruited locally become polarized toward M1-like pro-inflammatory macrophages, however during the resolution phase the macrophages transition to a more M2-like phenotype (19). At day 9, the peak of arthritis in the control mice, and a time when the Flipf/fLysMc/+ mice were clinically improving, an increased subset of F4/80hi macrophages was identified in the Flipf/fLysMc/+ mice. This population of macrophages was inversely correlated with the severity of the arthritis at the time of sacrifice. Further when cells were isolated from inflamed joints, the F4/80hi population expressed an increased M2-like phenotype, compared to the F4/80lo population of macrophages. These observations support the importance of macrophage repolarization as a therapeutic approach to inflammatory arthritis.

We initially intended to target Flip in myeloid cells as a means to therapeutically reduce synovial tissue macrophages by inducing apoptosis. Our earlier studies with human monocytes and macrophages demonstrated that FLIP was weakly expressed in monocytes, but highly expressed during monocyte to macrophage differentiation (5). Even though lysozyme M is expressed in monocytes (23), and the deleted Flip allele was seen in monocytes, the monocytes from Flipf/fLysMc/+ and control mice variably expressed FLIP. Consistent with these observations, there was no difference in the expression of Flip in the F4/80lo population in the Flipf/fLysMc/+ mice, compared with the controls. In contrast, the F4/80hi macrophages demonstrated a significant reduction of Flip in the Flipf/fLysMc/+ mice. This suggests that under inflammatory conditions, the requirement of FLIP for survival in macrophages may be circumvented. Supporting this interpretation there was no increase of apoptosis or necrosis of the F4/80hi population in Flipf/fLysMc/+ mice. Further, under inflammatory conditions bone marrow derived macrophages from Flipf/fLysMc/+ mice survived in the presence of reduced FLIP. These observations may be related to an increase of other anti-apoptotic molecules such as Mcl-1 or A20 which may also contribute to macrophage survival (24–27) and which are increased under inflammatory conditions.

A reduction of FLIP in surviving macrophages may have modulated intracellular signals increasing the differentiation toward F4/80hi, M2-like macrophages. Although caspase cleaved products of FLIP have been shown to activate NF-κB (28, 29), unprocessed FLIP may suppress NF-κB activation induced by LPS or Fas signaling (30, 31). Further the interaction of FLIP with the Fas-DISC while protecting against apoptosis recruits MyD88, suppressing TLR4 signaling (32), suggesting that the effects of FLIP may be context specific. FLIP also binds to MKK7 inhibiting JNK signaling (33), and it participates in activation of inflammasomes, and reduction of FLIP may be suppressive (34). FLIP is also capable of promoting Wnt signaling through reduction of β-catenin ubiquitination (35), which may modulate macrophage activation (36). FLIP may also suppress p38 activation in dendritic cells (37). Our data are consistent with the interpretation that under inflammatory conditions macrophages may survive despite the reduction of FLIP. Other studies suggest that not only inflammation, but also stress from hypoxia and energy deprivations, may protect cells from apoptosis (38). Therefore in macrophages capable of survival despite the reduction of FLIP under inflammatory conditions, the reduction of FLIP may promote an increase in the number of F4/80hi macrophages with an M2-like phenotype, potentially through alteration of intracellular signaling. Alternatively, in the control mice, the high expression FLIP may suppress this transition to F4/80hi. Together these observations suggest that under inflammatory conditions the reduction of FLIP in macrophages ameliorates STIA, not by reduction of macrophages but by permitting an increase of F4/80hi macrophages with an M2-like phenotype.

Supplementary Material

Supp Fig S1. Supplemental Figure 1. Characterization of the genotype and phenotype of Flipf/fLysMc/+ mice.

(A). PCR genotyping of single cell populations sorted from Flipf/fLysMc/+ mice were compared with Flipf/+LysMc/+ littermate controls. The LysM-cre driven specific deletion of floxed Flip band (Flipf) is identified by changing the 270 bp Flipf band into the 150 bp Flip deleted band (Flipd). The littermate control mice contain a wide type Flip allele (220 bp Flip+) which does not change in the presence of Cre recombinase. The single populations of cells were purified from peripheral blood by flow cytometry. (B). PCR of purified granulocytes from Flipf/fLysMc/+ mice was compared with those from Flipf/dLysMc/+ mice. (C). Body weight of adult mice. (D). Circulating monocytes and neutrophils, determined by complete blood counts, n > 50 for each group, 7–24 weeks of age. (E). Postnatal viability of Flipf/fLysMc/+, Flipf/dLysMc/+ and littermate controls was examined. Data are presented as the mean ±1 SE. Significance was analyzed by unpaired 2-sided t-test***represents p < 0.001.

Supp Fig S2. Supplemental Figure 2. Synovial granulocytes are increased on day 4 but not day 9 following STIA induction in Flipf/fLysMc/+ mice.

Granulocytes were identified by flow cytometry as CD45+CD11b+Ly6G+F4/80− on day 4 and day 9 post STIA induction (n= 5 each group at day4 and n= 7 for control and 8 for the Flipf/fLysMc/+ mice at day 9). Data are presented as the mean ± 1 SE, and significance was analyzed by unpaired 2-sided t-test, and * represents p <0.05.

Supp Fig S3. Supplemental Figure 3. FLIP is expressed in monocytes but not neutrophils.

FLIP expression in single cell populations was examined by Western blot. T cells were enriched from blood from one control and one Flipf/fLysMc/+ mouse as the FLIP expression control. Monocytes (CD115+CD11b+) and granulocytes (Ly6G+CD11b+) were isolated by flow cytometry, from the bone marrow (BM) or blood as indicated in the figure. For each of the controls, bone marrow from 2–3 mice were combined before sorting.

Acknowledgments

This study is supported by U.S. National Institutes of Heath Grants R21AR065076 (RMP), R01AR048269 (RMP), T32AR007611 (RMP), R01AR064546 (HP) and R01AR63650 (LX)

References

  • 1.Gierut A, Perlman H, Pope RM. Innate immunity and rheumatoid arthritis. Rheum Dis Clin North Am. 2010;36:271–96. doi: 10.1016/j.rdc.2010.03.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 2.McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med. 2011;365:2205–19. doi: 10.1056/NEJMra1004965. [DOI] [PubMed] [Google Scholar]
  • 3.Mulherin D, Fitzgerald O, Bresnihan B. Synovial tissue macrophage populations and articular damage in rheumatoid arthritis. Arthritis Rheum. 1996;39:115–24. doi: 10.1002/art.1780390116. [DOI] [PubMed] [Google Scholar]
  • 4.Wijbrandts CA, Vergunst CE, Haringman JJ, Gerlag DM, Smeets TJ, Tak PP. Absence of changes in the number of synovial sublining macrophages after ineffective treatment for rheumatoid arthritis: Implications for use of synovial sublining macrophages as a biomarker. Arthritis Rheum. 2007;56:3869–71. doi: 10.1002/art.22964. [DOI] [PubMed] [Google Scholar]
  • 5.Perlman H, Pagliari LJ, Georganas C, Mano T, Walsh K, Pope RM. FLICE-inhibitory protein expression during macrophage differentiation confers resistance to fas-mediated apoptosis. J Exp Med. 1999;190:1679–88. doi: 10.1084/jem.190.11.1679. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Perlman H, Pagliari LJ, Liu H, Koch AE, Haines GK, 3rd, Pope RM. Rheumatoid arthritis synovial macrophages express the Fas-associated death domain-like interleukin-1beta-converting enzyme-inhibitory protein and are refractory to Fas-mediated apoptosis. Arthritis Rheum. 2001;44:21–30. doi: 10.1002/1529-0131(200101)44:1<21::AID-ANR4>3.0.CO;2-8. [DOI] [PubMed] [Google Scholar]
  • 7.Okada Y, Wu D, Trynka G, Raj T, Chikashi T, Ikari K, et al. Genetics of rheumatoid arthritis contributes to biology and drug discovery. Nature. 2014;506:376–81. doi: 10.1038/nature12873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Huang QQ, Perlman H, Huang Z, Birkett R, Kan L, Agrawal H, et al. FLIP: a novel regulator of macrophage differentiation and granulocyte homeostasis. Blood. 2010;116:4968–77. doi: 10.1182/blood-2009-11-252841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Clausen BE, Burkhardt C, Reith W, Renkawitz R, Forster I. Conditional gene targeting in macrophages and granulocytes using LysMcre mice. Transgenic Res. 1999;8:265–77. doi: 10.1023/a:1008942828960. [DOI] [PubMed] [Google Scholar]
  • 10.Kouskoff V, Korganow AS, Duchatelle V, Degott C, Benoist C, Mathis D. Organ-specific disease provoked by systemic autoimmunity. Cell. 1996;87:811–22. doi: 10.1016/s0092-8674(00)81989-3. [DOI] [PubMed] [Google Scholar]
  • 11.Huang QQ, Birkett R, Koessler RE, Cuda CM, Haines G, 3rd, Jin JP, et al. Fas Signaling in Macrophages Promotes Chronicity in K/BxN Serum-Induced Arthritis. Arthritis Rheumatol. 2014;66:68–77. doi: 10.1002/art.38198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Scatizzi JC, Hutcheson J, Bickel E, Woods JM, Klosowska K, Moore TL, et al. p21Cip1 is required for the development of monocytes and their response to serum transfer-induced arthritis. Am J Pathol. 2006;168:1531–41. doi: 10.2353/ajpath.2006.050555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Huang QQ, Koessler RE, Birkett R, Dorfleutner A, Perlman H, Kenneth Haines G, 3rd, et al. Gp96 prpetuates the persistent inflammation of rheumatoid arthritis. Arthritis Rheum. 2012 doi: 10.1002/art.34610. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Huang QQ, Perlman H, Birkett R, Doyle R, Fang D, Haines GK, et al. CD11c-mediated deletion of Flip promotes autoreactivity and inflammatory arthritis. Nat Commun. 2015;6:7086. doi: 10.1038/ncomms8086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Zhang Q, Lu Y, Proulx ST, Guo R, Yao Z, Schwarz EM, et al. Increased lymphangiogenesis in joints of mice with inflammatory arthritis. Arthritis Res Ther. 2007;9:R118. doi: 10.1186/ar2326. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Zhang Q, Badell IR, Schwarz EM, Boulukos KE, Yao Z, Boyce BF, et al. Tumor necrosis factor prevents alendronate-induced osteoclast apoptosis in vivo by stimulating Bcl-xL expression through Ets-2. Arthritis Rheum. 2005;52:2708–18. doi: 10.1002/art.21236. [DOI] [PubMed] [Google Scholar]
  • 17.Haverkamp JM, Smith AM, Weinlich R, Dillon CP, Qualls JE, Neale G, et al. Myeloid-derived suppressor activity is mediated by monocytic lineages maintained by continuous inhibition of extrinsic and intrinsic death pathways. Immunity. 2014;41:947–59. doi: 10.1016/j.immuni.2014.10.020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Sheng J, Ruedl C, Karjalainen K. Most Tissue-Resident Macrophages Except Microglia Are Derived from Fetal Hematopoietic Stem Cells. Immunity. 2015;43:382–93. doi: 10.1016/j.immuni.2015.07.016. [DOI] [PubMed] [Google Scholar]
  • 19.Misharin AV, Cuda CM, Saber R, Turner JD, Gierut AK, Haines GK, 3rd, et al. Nonclassical Ly6C(−) monocytes drive the development of inflammatory arthritis in mice. Cell Rep. 2014;9:591–604. doi: 10.1016/j.celrep.2014.09.032. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Yona S, Kim KW, Wolf Y, Mildner A, Varol D, Breker M, et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity. 2013;38:79–91. doi: 10.1016/j.immuni.2012.12.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Gomez Perdiguero E, Klapproth K, Schulz C, Busch K, Azzoni E, Crozet L, et al. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature. 2015;518:547–51. doi: 10.1038/nature13989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Ginhoux F, Jung S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat Rev Immunol. 2014;14:392–404. doi: 10.1038/nri3671. [DOI] [PubMed] [Google Scholar]
  • 23.Eierman DF, Johnson CE, Haskill JS. Human monocyte inflammatory mediator gene expression is selectively regulated by adherence substrates. J Immunol. 1989;142:1970–6. [PubMed] [Google Scholar]
  • 24.Zhou P, Qian L, Bieszczad CK, Noelle R, Binder M, Levy NB, et al. Mcl-1 in transgenic mice promotes survival in a spectrum of hematopoietic cell types and immortalization in the myeloid lineage. Blood. 1998;92:3226–39. [PubMed] [Google Scholar]
  • 25.Tran TM, Temkin V, Shi B, Pagliari L, Daniel S, Ferran C, et al. TNFalpha-induced macrophage death via caspase-dependent and independent pathways. Apoptosis. 2009;14:320–32. doi: 10.1007/s10495-009-0311-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Liu H, Perlman H, Pagliari LJ, Pope RM. Constitutively activated Akt-1 is vital for the survival of human monocyte-differentiated macrophages. Role of Mcl-1, independent of nuclear factor (NF)-kappaB, Bad, or caspase activation. J Exp Med. 2001;194:113–26. doi: 10.1084/jem.194.2.113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Li HL, Wang AB, Zhang R, Wei YS, Chen HZ, She ZG, et al. A20 inhibits oxidized low-density lipoprotein-induced apoptosis through negative Fas/Fas ligand-dependent activation of caspase-8 and mitochondrial pathways in murine RAW264.7 macrophages. J Cell Physiol. 2006;208:307–18. doi: 10.1002/jcp.20665. [DOI] [PubMed] [Google Scholar]
  • 28.Golks A, Brenner D, Krammer PH, Lavrik IN. The c-FLIP-NH2 terminus (p22-FLIP) induces NF-kappaB activation. J Exp Med. 2006;203:1295–305. doi: 10.1084/jem.20051556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Kataoka T, Budd RC, Holler N, Thome M, Martinon F, Irmler M, et al. The caspase-8 inhibitor FLIP promotes activation of NF-kappaB and Erk signaling pathways. Curr Biol. 2000;10:640–8. doi: 10.1016/s0960-9822(00)00512-1. [DOI] [PubMed] [Google Scholar]
  • 30.Bannerman DD, Eiting KT, Winn RK, Harlan JM. FLICE-like inhibitory protein (FLIP) protects against apoptosis and suppresses NF-kappaB activation induced by bacterial lipopolysaccharide. Am J Pathol. 2004;165:1423–31. doi: 10.1016/s0002-9440(10)63400-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Kreuz S, Siegmund D, Rumpf JJ, Samel D, Leverkus M, Janssen O, et al. NFkappaB activation by Fas is mediated through FADD, caspase-8, and RIP and is inhibited by FLIP. J Cell Biol. 2004;166:369–80. doi: 10.1083/jcb.200401036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Ma Y, Liu H, Tu-Rapp H, Thiesen HJ, Ibrahim SM, Cole SM, et al. Fas ligation on macrophages enhances IL-1R1-Toll-like receptor 4 signaling and promotes chronic inflammation. Nat Immunol. 2004;5:380–7. doi: 10.1038/ni1054. [DOI] [PubMed] [Google Scholar]
  • 33.Nakajima A, Komazawa-Sakon S, Takekawa M, Sasazuki T, Yeh WC, Yagita H, et al. An antiapoptotic protein, c-FLIPL, directly binds to MKK7 and inhibits the JNK pathway. EMBO J. 2006;25:5549–59. doi: 10.1038/sj.emboj.7601423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Wu YH, Kuo WC, Wu YJ, Yang KT, Chen ST, Jiang ST, et al. Participation of c-FLIP in NLRP3 and AIM2 inflammasome activation. Cell Death Differ. 2014;21:451–61. doi: 10.1038/cdd.2013.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Naito M, Katayama R, Ishioka T, Suga A, Takubo K, Nanjo M, et al. Cellular FLIP inhibits beta-catenin ubiquitylation and enhances Wnt signaling. Mol Cell Biol. 2004;24:8418–27. doi: 10.1128/MCB.24.19.8418-8427.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Schaale K, Neumann J, Schneider D, Ehlers S, Reiling N. Wnt signaling in macrophages: augmenting and inhibiting mycobacteria-induced inflammatory responses. Eur J Cell Biol. 2011;90:553–9. doi: 10.1016/j.ejcb.2010.11.004. [DOI] [PubMed] [Google Scholar]
  • 37.Wu YJ, Wu YH, Mo ST, Hsiao HW, He YW, Lai MZ. Cellular FLIP Inhibits Myeloid Cell Activation by Suppressing Selective Innate Signaling. J Immunol. 2015;195:2612–23. doi: 10.4049/jimmunol.1402944. [DOI] [PubMed] [Google Scholar]
  • 38.Milisav I, Poljsak B, Ribaric S. Reduced risk of apoptosis: mechanisms of stress responses. Apoptosis. 2017;22:265–83. doi: 10.1007/s10495-016-1317-3. [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supp Fig S1. Supplemental Figure 1. Characterization of the genotype and phenotype of Flipf/fLysMc/+ mice.

(A). PCR genotyping of single cell populations sorted from Flipf/fLysMc/+ mice were compared with Flipf/+LysMc/+ littermate controls. The LysM-cre driven specific deletion of floxed Flip band (Flipf) is identified by changing the 270 bp Flipf band into the 150 bp Flip deleted band (Flipd). The littermate control mice contain a wide type Flip allele (220 bp Flip+) which does not change in the presence of Cre recombinase. The single populations of cells were purified from peripheral blood by flow cytometry. (B). PCR of purified granulocytes from Flipf/fLysMc/+ mice was compared with those from Flipf/dLysMc/+ mice. (C). Body weight of adult mice. (D). Circulating monocytes and neutrophils, determined by complete blood counts, n > 50 for each group, 7–24 weeks of age. (E). Postnatal viability of Flipf/fLysMc/+, Flipf/dLysMc/+ and littermate controls was examined. Data are presented as the mean ±1 SE. Significance was analyzed by unpaired 2-sided t-test***represents p < 0.001.

NIHMS875710-supplement-Supp_Fig_S1.tif (2.4MB, tif)
Supp Fig S2. Supplemental Figure 2. Synovial granulocytes are increased on day 4 but not day 9 following STIA induction in Flipf/fLysMc/+ mice.

Granulocytes were identified by flow cytometry as CD45+CD11b+Ly6G+F4/80− on day 4 and day 9 post STIA induction (n= 5 each group at day4 and n= 7 for control and 8 for the Flipf/fLysMc/+ mice at day 9). Data are presented as the mean ± 1 SE, and significance was analyzed by unpaired 2-sided t-test, and * represents p <0.05.

NIHMS875710-supplement-Supp_Fig_S2.tif (892.3KB, tif)
Supp Fig S3. Supplemental Figure 3. FLIP is expressed in monocytes but not neutrophils.

FLIP expression in single cell populations was examined by Western blot. T cells were enriched from blood from one control and one Flipf/fLysMc/+ mouse as the FLIP expression control. Monocytes (CD115+CD11b+) and granulocytes (Ly6G+CD11b+) were isolated by flow cytometry, from the bone marrow (BM) or blood as indicated in the figure. For each of the controls, bone marrow from 2–3 mice were combined before sorting.

NIHMS875710-supplement-Supp_Fig_S3.tif (319.7KB, tif)

RESOURCES