Monocytes are derived from bone marrow (BM) macrophage (Mϕ) and dendritic cell progenitor cells (MDPs), which develop into common monocyte progenitor cells (cMoPs) and then Ccr2⁺Ccr4^loLy6C⁺ monocytes. Ccl2, a ligand for Ccr2, is essential for the egress of Ly6C⁺ monocytes from the BM in response to inflammation (Serbina and Pamer, 2006; Hanna et al., 2011), after which these cells can develop into Ly6C^hiCcr2^hiCx3cr1^loCD43⁻ (Ly6C^hi) ‘classical’ monocytes or Ly6C^lo/−Ccr2^loCx3cr1^hiCD43⁺ (Ly6C⁻) ‘non-classical’ monocytes (Yona et al., 2013). CD62L (Sell), a marker expressed by newly emigrated BM-derived Ly6C⁺ monocytes, is lost with maturation (Ingersoll et al., 2010; Jakubzick et al., 2017).

Ly6C⁻ monocytes are derived from Ly6C^hi precursors (Yona et al., 2013; Sunderkötter et al., 2004; Varol et al., 2007; Sugimoto et al., 2015). In unmanipulated C57BL/6 (B6) mice there are two populations of circulating monocytes: Ly6C^hi (expressing Ccr2, Lyz2, Sell, Irf4, and other genes) and Ly6C⁻ (expressing Cx3cr1, Itgax, Bcl2, Pparg, Cd36, Cebpb, Nr4a1, and other genes). A third population with intermediate expression of Ly6C (referred to here as Ly6C^lo) does not have a specific gene expression signature, but expresses genes characteristic of both Ly6C^hi and Ly6C⁻ monocytes (Mildner et al., 2017).

Differentiation of Ly6C⁻ monocytes is driven by CCAAT-enhancer binding protein β (C/EBPβ) via activation of the transcription factor nuclear receptor subfamily 4 group A member 1 (Nr4a1) (Hanna et al., 2011; Mildner et al., 2017). Along with Nr4a1, Ly6C⁻ monocyte development requires Notch signaling (Hanna et al., 2011; Gamrekelashvili et al., 2016). In contrast to the role of Ly6C^hi monocytes as mediators of inflammation, Nr4a1-dependent Ly6C⁻ monocytes patrol the vascular endothelium and promote the TLR7-dependent removal of damaged cells (Auffray et al., 2007; Carlin et al., 2013). Although they typically remain within blood vessels, they also can migrate across the endothelium into inflamed tissues (Auffray et al., 2007; Schyns et al., 2019). Ly6C⁻ monocytes may represent a form of intravascular macrophage (Mϕ) (Ginhoux and Jung, 2014), as suggested by the expression of genes typical of tissue-resident Mϕ such as Apoe and Cd36 (Mildner et al., 2017). Although only a single population of Ly6C⁻ monocytes is seen in the blood of unmanipulated B6 mice, less is known about Ly6C⁻ monocytes in inflammatory settings.

Pristane and mineral oil (MO) both cause sterile peritoneal inflammation and an influx of Ly6C^hi monocytes, which become Ly6C^hi inflammatory peritoneal Mϕ (Lee et al., 2009). In pristane—but not MO—treated mice, the chronic inflammatory response evolves into lupus after several months (Reeves et al., 2009). In pristane-treated B6 mice, but not BALB/c or other strains, the onset of lupus is complicated by lung microvascular injury and diffuse alveolar hemorrhage (DAH) (Zhuang et al., 2017; H Zhuang, In Revision).

In contrast to the striking predominance of Ly6C^hi Mϕ in the peritoneum of pristane-treated mice, Ly6C^lo Mϕ predominate in MO-treated mice (Han et al., 2017). A subset of the Ly6C⁻ peritoneal Mϕ expresses CD138 (syndecan-1) (Han et al., 2017). Ly6C^loCD138⁺ Mϕ from MO-treated mice have an anti-inflammatory phenotype and are highly phagocytic for dead cells. The significance of CD138 expression by Mϕ is unclear and it is not known whether CD138 is expressed by monocytes. We examined the distribution of CD138 expression in myeloid cells from the blood and inflamed peritoneum of pristane- and MO-treated mice.

Peritoneal Ly6C^loCD138⁺ and Ly6C^loCD138⁻ Mϕ both were Nr4a1 independent and Ccr2 dependent, suggesting they were derived from classical (Ly6C^hi) monocytes recruited to the peritoneum. Unexpectedly, in pristane-treated mice circulating Ly6C^−/lo monocytes also consisted of CD138⁺ and CD138⁻ populations. However, only the Ly6C^−/loCD138⁺ subset was Nr4a1 dependent. Compared with the Ly6C^−/loCD138⁻ and Ly6C^hi subsets, Ly6C^−/loCD138⁺ monocytes expressed higher levels of Triggering Receptor Expressed on Myeloid Cells Like 4 (Treml4), an innate immune receptor that amplifies TLR7 signaling and binds late apoptotic and necrotic cells (Hemmi et al., 2009; Ramirez-Ortiz et al., 2015; Nedeva et al., 2020; Gonzalez-Cotto et al., 2020). Treml4 expression was regulated by Nr4a1, consistent with a role in the Ly6C⁻ monocyte-mediated, TLR7-dependent, removal of damaged endothelial cells. Circulating Nr4a1-dependent Ly6C⁻CD138⁺ monocytes increased substantially after pristane, but not MO, treatment, and were more abundant in B6 (DAH susceptible) vs. BALB/c (DAH resistant) mice. In contrast to pristane-treated mice, Nr4a1-dependent Ly6C^lo/− monocytes in MO-treated mice were CD138⁻. Thus, Nr4a1-dependent Ly6C^−/lo ‘patrolling’ monocytes from pristane- vs. MO-treated mice were distinguishable by the presence or absence, respectively, of CD138 surface staining. Monocytes (both Ly6C^hi and Ly6C^−/lo) from pristane- vs. MO-treated also were distinguished by the intensity of annexin-V staining, suggesting that pristane treatment causes a breakdown of plasma membrane asymmetry, which could have functional implications for the pathogenesis of pristane-induced lupus and alveolar hemorrhage.

Although i.p. pristane injection induces lupus autoantibodies and renal disease in both B6 and BALB/c mice, only B6 mice develop DAH (Reeves et al., 2009). MO causes peritoneal inflammation in both strains, but lupus does not develop. Pristane-induced DAH is abolished by depleting monocytes and Mϕ with clodronate liposomes, whereas neutrophil depletion has little effect (Zhuang et al., 2017), suggesting that it is mediated at least partly by monocytes and/or Mϕ. There is little information about monocytes in pristane-treated mice and their relationship to disease pathogenesis. Pristane-induced Ly6C^hi monocyte recruitment from the BM to the peritoneum is Ccr2 dependent (Lee et al., 2009). A population of highly phagocytic peritoneal Ly6C⁻CD138⁺ Mϕ with an anti-inflammatory phenotype is more abundant in MO- vs. pristane-treated mice, whereas Ly6C^hiCD138⁻ Mϕ are more abundant in pristane-treated mice (Han et al., 2017). We examined the origin of these peritoneal Ly6C⁻CD138⁺ Mϕ.

Circulating CD138⁺ monocytes

Ly6C^hi monocytes recruited to the inflamed peritoneum become Ly6C^hi Mϕ, which subsequently can downregulate Ly6C and develop into Ly6C^−/lo Mϕ (Lee et al., 2009). Ly6C^hi monocytes also give rise to Ly6C^−/lo monocytes in the circulation (Sunderkötter et al., 2004; Mildner et al., 2017), raising the question of whether peritoneal Ly6C^−/loCD138⁺ Mϕ are derived from circulating Ly6C^−/lo monocytes or from peritoneal Ly6C^hi Mϕ. CD138⁺CD11b⁺ cells were found in the blood of both pristane- and MO-treated B6 mice at 14 days, but were considerably more abundant in pristane-treated mice (Figure 1A). In contrast, peritoneal CD138⁺CD11b⁺ Mϕ were more abundant in MO-treated mice than pristane-treated mice. BALB/c mice and B6 mice lacking B cells (μMT) do not develop pristane-induced DAH (Zhuang et al., 2017; Barker et al., 2011). Like MO-treated B6 mice, both B6 μMT and BALB/c mice generally had fewer circulating CD138⁺CD11b⁺ cells and more peritoneal CD138⁺CD11b⁺ Mϕ than wild-type B6 mice (Figure 1B). These observations suggested that circulating CD138⁺CD11b⁺ myeloid cells were recruited to the peritoneum and developed into CD138⁺CD11b⁺ Mϕ or alternatively, that CD138⁺CD11b⁺ Mϕ were generated locally in the peritoneum. As an initial step to distinguish between these possibilities, we carried out more extensive phenotyping of the circulating and peritoneal populations.

Figure 1

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Flow cytometry revealed three subsets of CD11b⁺Ly6G⁻ cells from pristane-treated B6 mice in both the blood and the peritoneum (Figure 2A): CD11b⁺Ly6C^hiCD138⁻ (R1); CD11b⁺Ly6C^−/loCD138⁻ (R2); and CD11b⁺Ly6C^−/loCD138⁺ (R3). Ly6C staining was higher on the peritoneal R2/R3 cells (Ly6C^lo) vs. blood R2/R3 (Ly6C⁻) cells. In the blood of pristane-treated mice, all three subsets were CD43⁺ and TremL4⁺, consistent with a monocyte phenotype (Figure 2B,C). The blood R3 and R2 subsets expressed higher levels of CD43 and TremL4 than R1; R3 expressed higher levels than R2. In the blood CD115 was expressed at low levels, mainly on R3 cells (Figure 2D). Interestingly, CD43, TremL4, and CD115 were highly expressed by the peritoneal R3, and to a lesser extent R2, subsets from MO-treated, but not pristane-treated, mice (Figure 2B–D). As expected, surface staining for CD62L, which is expressed by Ly6C^hi monocytes that have recently migrated out of the BM and not on Ly6C^lo monocytes (Jakubzick et al., 2017), was stronger in R1 vs. R3 or R2 (Figure 2E). Expression was highest in the circulating R1 subset. As classical monocytes are CD115⁺Ly6C⁺CD43^lo whereas non-classical monocytes are CD115⁺Ly6C⁻CD43^hiTremL4⁺ (Jakubzick et al., 2017), we concluded that the circulating R1 cells were classical (Ly6C^hi) monocytes and the circulating R2 and R3 cells were subsets of non-classical monocytes. Circulating R3 cells also were CX3CR1⁺CCR2⁻ (Figure 2F,G).

Figure 2

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Murine non-classical monocytes differentiate in the circulation from Ly6C^hi precursors, have a Ly6C⁻CD43⁺CX3CR1^hi phenotype and may represent terminally differentiated blood-resident Mϕ rather than true monocytes (Ginhoux and Jung, 2014). Consistent with that possibility, circulating R3 monocytes from pristane-treated mice expressed higher levels of the Mϕ marker F4/80 than R1 or R2 monocytes (Figure 2H). However, another Mϕ marker (CD64, FcγR1a) was expressed at lower levels on R3 (and R2) vs. R1 (Figure 2I). Peritoneal R3 Mϕ stained more intensely for F4/80, CD64, CCR2, and CX3CR1 than circulating R3 monocytes, and with the exception of CX3CR1, exhibited stronger staining than peritoneal R2 Mϕ. Together the data suggest that blood of pristane-treated mice contains classical monocytes along with two subtypes of non-classical monocytes distinguished by the presence (R3) or absence (R2) of CD138 staining. Peritoneal Mϕ also consisted of R1, R2, and R3 populations. Circulating R3 cells had a phenotype compatible with that of ‘patrolling’ monocytes but also had higher F4/80 staining than circulating R1 or R2 cells, consistent with early Mϕ differentiation. To further characterize the origin of this unusual monocyte subset, we examined CD138 monocyte development in Ccr2−/− and Nr4a1−/− mice.

CD138⁺ monocytes in Ccr2−/− mice

Monocyte egress from the BM is defective in Ccr2-deficient mice (Serbina and Pamer, 2006) and Ccr2 is necessary for Ly6C^hi monocytes to migrate to the peritoneum in pristane-treated mice (Lee et al., 2009). After Ly6C^hi (R1) monocytes migrate to the peritoneum, they can downregulate Ly6C. It is not known whether both Ly6C^lo peritoneal subsets (CD138⁺, R3 and CD138⁻, R2) are generated from Ly6C^hi precursors or if the peritoneal R3 subset is derived from circulating CD138⁺ monocytes. Circulating Ly6C^hi (R1) monocytes were strongly Ccr2⁺, whereas Ly6C⁻CD138⁻ (R2) and Ly6C⁻CD138⁺ (R3) monocytes expressed low levels (Figure 2F). In contrast, Ccr2 was expressed by all peritoneal subsets in pristane-treated mice, most strongly by R3. Ccr2 also was expressed by R1, R2, and R3 from MO-treated mice, but at lower levels (Figure 2F). Staining of circulating monocytes for Cx3Cr1, which is expressed at high levels on Ly6C⁻ monocytes but is involved in homing of both Ly6C^hi and Ly6C⁻ monocytes (Tacke et al., 2007; Landsman et al., 2009), was weak in pristane-treated mice, whereas peritoneal cells (both R2 and R3) expressed higher levels (Figure 2G). Expression was higher on peritoneal Mϕ from pristane- vs. MO-treated mice.

Circulating monocyte subsets varied with time after pristane treatment (Figure 3A). Ly6C^hi monocytes (R1) increased first, about 5 days after pristane or MO treatment, while at the same time, the percentage of Ly6C⁻CD138⁻ monocytes (R2) decreased. Circulating Ly6C⁻CD138⁺ monocytes (R3) appeared in pristane-treated mice at day 9 but only small numbers were seen in MO-treated mice (Figure 3A).

Figure 3

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As expected, circulating R1 monocytes were greatly reduced (but not absent) in pristane-treated Ccr2−/− mice (Figure 3B). R3 monocytes also were greatly reduced in Ccr2−/− mice, suggesting that, like R1 monocytes, their migration from the BM to the circulation was Ccr2 dependent. In contrast to R1 and R3, the percentages of circulating R2 cells were similar in Ccr2−/− vs. wild-type mice. In PECs, R1 and R3 cells were absent and R2 cells were present at lower levels in Ccr2−/− mice vs. wild-type (Figure 3B). Thus, the egress of R1 (Ly6C^hi) monocytes from the BM and the appearance of R3 (Ly6C⁻CD138⁺) monocytes in the circulation was Ccr2 dependent, whereas the circulating R2 monocyte subset (Ly6C⁻CD138⁻) was unaffected by the absence of Ccr2. R1 and R3 monocytes appeared in the circulation with different kinetics and all three peritoneal Mϕ subsets were reduced in Ccr2−/− mice, consistent with the possibility that they were derived from CCR2⁺ R1 monocytes.

Nr4a1−/− mice lack CD138⁺ monocytes but not CD138⁺ peritoneal Mϕ

Nr4a1 (Nur77) controls the differentiation of Ly6C⁻ monocytes (Hanna et al., 2011) and regulates inflammatory responses in Mϕ (Hanna et al., 2012). Peripheral blood cells and PECs from wild-type and Nr4a1−/− mice were analyzed by flow cytometry 14 days after pristane or MO treatment, gating on forward/side scatter and surface staining characteristics (Figure 4A). As expected (Han et al., 2017), most peritoneal CD11b⁺Ly6G⁻ cells in MO-treated mice were Ly6C^lo and many were CD138⁺ (Figure 4A). After pristane treatment, percentages of Ly6C^hi (R1), Ly6C^loCD138⁻ (R2), and Ly6C^loCD138⁺ (R3) peritoneal Mϕ were similar in Nr4a1−/− and wild-type mice (Figure 4A,B). In contrast, although the percentages of circulating R1 and R2 monocytes were similar, Nr4a1−/− mice had a markedly lower percentage of circulating CD138⁺ (R3) monocytes than wild-type mice (Figure 4C).

Figure 4

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Along with CD11b⁺Tim4⁺ resident (yolk sac-derived) ‘large peritoneal macrophages’ (LPMs) (Ghosn et al., 2010), the uninflamed peritoneum contains CD11b⁺Tim4⁻CD138⁺ cells, which are probably ‘small peritoneal macrophages’ (SPMs) (Ghosn et al., 2010). The percentages of resident LPM in untreated wild-type vs. Nr4a1−/− mice were similar, whereas the percentage was higher in Ccr2−/− mice (Figure 4D), probably reflecting the absence of BM-derived cells. The CD138⁺ SPM subset was increased in Nr4a1−/− mice vs. wild-type and Ccr2−/− mice, suggesting that they originated from circulating BM-derived (Nr4a1-independent) Ly6C⁺ monocytes rather than Nr4a1-dependent CD138⁺ monocytes.

Phenotypes of circulating Ly6C^lo monocytes

Although non-classical monocytes are characteristically Ly6C⁻, CD138 expression has not been reported. We carried out more extensive phenotyping to better define the unusual (CD138⁺) R3 subset. Gating on CD11b⁺Ly6G⁻ (non-neutrophil) peripheral blood cells (mainly monocytes), R2 and R3 could be divided into Ly6C^lo and Ly6C⁻ subsets. Five groups of circulating myeloid (non-neutrophil) cells could be distinguished: Ly6C^hiCD138⁻ (R1), Ly6C^loCD138⁻ (R2A), Ly6C⁻CD138⁻ (R2B), Ly6C^loCD138⁺ (R3A), and Ly6C⁻CD138⁺ (R3B) (Figure 5A). All five subsets were present in pristane-treated B6 mice, whereas only R1, R2A, and R2B were present in phosphate-buffered saline (PBS)- or MO-treated mice. Pristane-treated Nr4a1−/− mice showed a pattern similar to that of MO-treated wild-type mice, though R3A and R3B cells were somewhat more abundant (Figure 5A). CD138 was not expressed on CD11b⁺Ly6G⁻ cells from PBS- or MO-treated mice (Figure 5A), so Ly6C^−/lo cells in pristane-treated mice consisted of R2A, R2B, R3A, and R3B, whereas those from PBS- or MO-treated mice were almost exclusively R2A and R2B (Figure 5B). Nr4a1−/− mice had low percentages of circulating CD138⁺ R3A and R3B monocytes vs. wild-type mice, but the percentages were higher than those in MO- or PBS-treated mice (Figure 5B, right).

Figure 5

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CD138 staining (mean fluorescence intensity, MFI) was much higher in R3A and R3B than in R2A and R2B (Figure 5C). CD11b staining intensity showed a similar trend: higher in R3A/R3B vs. R2A/R2B. Since DAH is monocyte dependent and does not develop in pristane-treated mice lacking CR3 [Itgam−/− (CD11b) or Itgb2−/− (CD18) mice] (Zhuang et al., 2017; Shi et al., 2014), we also analyzed circulating monocytes based on Ly6C and CD11b staining. Similar to Ly6C/CD138 staining, five monocyte subsets were defined in pristane-treated mice based on Ly6C and CD11b staining: R4 (Ly6C^hiCD11b^+/hi), R5A (Ly6C^loCD11b⁺), R5B (Ly6C⁻CD11b⁺), R6A (Ly6C^loCD11b^hi), and R6B (Ly6C⁻CD11b^hi) (Figure 5C vs. Figure 5D ). CD138 and CD11b staining were both higher on (R6A/R6B) than on R5A/R5B, suggesting that the two subsets of CD138⁺ monocytes are Ly6C^loCD138⁺CD11b^hi and Ly6C⁻CD138⁺CD11b^hi, respectively.

Further characterization of circulating monocytes from pristane-treated mice revealed that CD62L was expressed at high levels (as expected) on R1 and R4 cells (Figure 6A). CD62L was absent on R3A/R3B and R6A/R6B cells, and was expressed at intermediate levels on R2A/R2B and R5A/R5B (Figure 6A). Nr4a1, a transcription factor critical for generating non-classical monocytes (Hanna et al., 2011), was expressed intracellularly at high levels in R3A/R3B (and R6A/R6B), low levels in R1 (and R4), and intermediate levels in R2A/R2B (and R5B) (Figure 6B). However, levels of Nr4a1 were low in R5A. Ly6C⁻LFA1^hiCCR2^loCX3CR1^hi patrolling monocytes adhere and crawl along the endothelial cells in postcapillary venules, a process that is LFA-1 dependent (Auffray et al., 2007). Cx3Cl1 (fractalkine), the ligand for Cx3Cr1, is expressed on endothelial cells and mediates adhesion of monocytes to endothelial cells, whereas blocking antibodies to LFA-1 can detach crawling monocytes in Cx3cr1+/− mice (Auffray et al., 2007). Consistent with the possibility that they are patrolling monocytes, Cx3Cr1 and LFA-1 were expressed more highly by R3A/R3B (and R6A/R6B) monocytes than by the other subsets (Figure 6C,D). Expression of CD11c, a differentiation marker expressed by dendritic cells as well as circulating ‘foamy monocytes’ containing intracellular lipid droplets (Wu et al., 2009; Xu et al., 2015), was greatly increased on the R3B (R6B) subset (Figure 6E). Phenotypes of the R1, R2A/R2B, and R3A/R3B subsets are summarized in Table 1.

Figure 6

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Table 1

	Ly6C	CD138	CD11b	Nr4a1	CCR2	Cx3Cr1	CD62L	CD11c	LFA1	Treml4	TLR7	TNFa	CD43	CD115
R1 (P)*	++	(−)	+	(−)	++	+	++	(−)	+	(−)	+	++	++	+
R2A (P)	+	(−)	+	+	+	+	+	+	++	+	+	++	+	+
R2B (P)	(−)	(−)	+	+	(−)	+	+	+	++	+	+	+	+	+
R3A (P)	+	++	++	++	+	++	(−)	+	++	++	++	++	+	++
R3B (P)	(−)	++	+	+	(−)	++	(−)	++	++	++	++	+	++	++
R1 (MO)*	++	(−)	+	(−)	n.d.	n.d.	n.d.	(−)	n.d.	(−)	+	+	+	n.d.
R3A (MO)	+	(−)	++	++	n.d.	n.d.	n.d.	+	n.d.	++	+	++	+	n.d.
R3B (MO)	(−)	(−)	+	+	n.d.	n.d.	n.d.	++	n.d.	++	+	+	+	n.d.

1. *P, pristane; MO, mineral oil; n.d., not determined.

TLR7 ligand-stimulated TNFα production by Ly6C⁻CD138⁺ monocytes

Phenotypic analysis suggested that the Ly6C⁻CD138⁺ subset may be a variant of patrolling monocytes, which encounter damaged endothelial cells respond to danger signals via TLR7 and then migrate into tissues where they undergo cMaf/MafB-regulated Mϕ differentiation and produce TNFα (Auffray et al., 2007; Ginhoux and Jung, 2014). Ly6C⁻ cells are thought to promote focal endothelial necrosis followed by repair. We hypothesized that Ly6C⁻CD138⁺ monocytes might respond more aggressively to endothelial injury, potentially exacerbating damage rather than promoting resolution/repair. Along with their acquisition of the dendritic cell marker CD11c (Figure 6E), the R3A/R3B (and R6A/R6B) subsets from pristane-treated B6 mice expressed considerably higher levels of TLR7 than other circulating monocyte subsets (Figure 7A). These cells also expressed high levels of Treml4 (Figure 7B), a protein that amplifies TLR7 signaling (Ramirez-Ortiz et al., 2015). As shown in Figure 7C, TLR7 ligand (R848)-stimulated intracellular TNFα staining was comparable in R3 monocytes vs. ‘inflammatory’ Ly6C^hi monocytes. Moreover, TNFα staining was significantly higher in Ly6C⁻CD138⁺ (R3) monocytes vs. Ly6C⁻CD138⁻ (R2) monocytes (Figure 7C, right).

Figure 7

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To investigate whether monocytes from pristane-treated mice were more prone to undergo apoptosis in response to TLR7 signaling, blood from 14-day pristane- or MO-treated mice was incubated for 20 hr with R848 (1 μg/ml) or PBS and then stained with anti-CD11b, Ly6C, and Ly6G antibodies plus PE-conjugated annexin-V. As shown in Figure 7D (left), the MFI of annexin-V staining was higher on Ly6C^hi and Ly6C^lo/− monocytes from pristane- vs. MO-treated mice. The MFI of neutrophils from pristane- vs. MO-treated mice was similar. Addition of R848 to the cultures did not significantly change the intensity of annexin-V staining. Histograms revealed that monocyte annexin-V staining was uniformly shifted to the right in pristane- vs. MO-treated mice, but there was only a single peak. In contrast, in neutrophils a second peak with more intense staining, most likely representing apoptotic cells, was seen, more prominently in pristane- vs. MO-treated mice (Figure 7D, right, arrows). Monocytes did not exhibit this second peak. Annexin-V staining also was shifted to the right in freshly isolated monocytes (without in vitro culture) from pristane- vs. MO-treated mice (not shown). Intracellular staining of freshly isolated cells with anti-activated caspase-3 antibodies was negative (not shown), suggesting that the annexin-V⁺ monocytes from pristane-treated mice were not apoptotic.

Low TremL4 expression in Nr4a1−/− mice

TremL4 amplifies proinflammatory signaling through TLR7 by regulating the recruitment of MyD88 (Hemmi et al., 2009; Ramirez-Ortiz et al., 2015; Nedeva et al., 2020; Gonzalez-Cotto et al., 2020). It is expressed by Ly6C⁻ monocytes and Nr4a1⁺Ly6C^hi monocytes committed to differentiate into Ly6C⁻ monocytes (Briseño et al., 2016). Circulating CD11b⁺Treml4⁺ monocytes increased in pristane-treated wild-type mice, and to a lesser degree in untreated wild-type mice vs. pristane-treated Nr4a1−/− mice (Figure 8A). In both wild-type and Nr4a1−/− mice Treml4 staining intensity was highest in circulating CD138⁺Ly6C⁻ monocytes (R3) and was lower in CD138⁻Ly6C^−/lo (R2A/R2B) monocytes (Figure 8B). R1 cells were only weakly positive and neutrophils did not stain for TremL4.

Figure 8

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Nearly all circulating CD138⁺ (R3) cells were TremL4⁺ (Figure 8C, boxes). There was a suggestion that Ly6C^hi (R1) cells may acquire Treml4 surface staining concomitantly with downregulation of Ly6C and upregulation of CD138 (Figure 8C, arrows, lower right). The acquisition of TremL4 and CD138 staining was attenuated in Nr4a1−/− mice. In contrast, although signaling through the type I interferon receptor (Ifnar1) blocks the downregulation of Ly6C in peritoneal Mϕ, Ifnar1−/− and wild-type mice had similar numbers of circulating CD138⁺TremL4⁺ monocytes (Figure 8C). Mean Nr4a1 staining intensity in total CD11b⁺Ly6G⁻ cells from pristane- and PBS-treated mice correlated with the percentage of circulating TremL4⁺CD11b⁺Ly6G⁻ cells (Figure 8D), suggesting that Nr4a1 might regulate TremL4. Consistent with that possibility, pristane and MO treatment both increased the fluorescence intensity of Nr4a1 and TremL4 in circulating CD11b⁺Ly6G⁻ cells (Figure 8E).

Ly6C⁻CD138⁺ monocytes are associated with DAH

Circulating (Nr4a1-dependent) Ly6C⁻CD138⁺ monocytes were present in pristane-treated mice with DAH, but not MO-treated mice without DAH. However, Nr4a1−/− mice were susceptible to the induction of DAH by pristane (Figure 4E). Circulating Ly6C^-CD138⁺ monocytes were substantially lower in BALB/c (resistant to DAH) vs. B6 (DAH-susceptible) mice and there was a similar trend in pristane-treated B-cell-deficient (μMT) B6 mice, which also fail to develop DAH (Zhuang et al., 2017; Figure 1B). Intracellular Nr4a1 staining (flow cytometry) was lower in circulating Ly6C^hi and Ly6C^lo monocytes from untreated BALB/c vs. B6 mice (Figure 9A). Thus, increased numbers of circulating, Nr4a1-dependent, Ly6C^loCD138⁺ monocytes and a high level of monocyte Nr4a1 protein expression were associated with susceptibility to DAH.

Figure 9

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Although Nr4a1-dependent Ly6C⁻ patrolling monocytes are present in untreated B6 mice, they did not express CD138 in RNA-Seq studies (Mildner et al., 2017). There were few CD138⁺Ly6C⁻ monocytes in either untreated or MO-treated mice (Figures 1 and 5A). In MO-treated mice, CD138⁻Ly6C⁻ monocytes were present in the circulation (Figure 5A), but they were not reduced in Nr4a1−/− mice (Figures 4C and 5A). We hypothesized that MO-treated mice may have Nr4a1-dependent Ly6C^−/loCD138⁻ (R2) monocytes equivalent to the R3 subset in pristane-treated mice. In view of the extremely low number of Ly6C^−/loCD138⁺ cells in MO-treated or Nr4a1−/− mice (Figure 4 and Figure 5), we examined Ly6C^−/loCD11b^hi monocytes (Figure 5C,D), which are likely to be similar to the Ly6C^−/loCD138⁺ subset (Figure 6). Significant numbers of circulating Ly6C^−/loCD11b^hi (R6A/R6B) monocytes were seen in MO-treated wild-type mice and Nr4a1−/− mice (Figure 9B,C). R6A cells were higher in Nr4a1−/− mice vs. wild type, whereas R6B cells were significantly lower in Nr4a1−/− mice. R5B cells also were reduced in Nr4a1−/− mice (Figure 9C). Thus, the development in MO-treated mice of Ly6C⁻CD11b^hi (R6B) and Ly6C⁻CD11b⁺ (R5B) monocytes, like the development of Ly6C⁻CD138⁺ (R3B) monocytes in pristane-treated mice, was Nr4a1 dependent. Together, the data suggest the following: (1) Nr4a1-dependent Ly6C⁻ monocytes express CD138 in pristane-treated mice that develop DAH, but not in mice that are resistant to pristane-induced DAH (MO-treated B6, B6 μMT, and BALB/c) and (2) the R5B and R6B subsets in MO-treated mice are Nr4a1 regulated.

Surface staining of blood cells from MO-treated mice showed high levels of TremL4 expression in the R5B, R6A, and R6B subsets (Figure 9D). The expression of TremL4 was reduced substantially in Nr4a1−/− mice, further suggesting that TremL4 might be Nr4a1 regulated. This possibility was examined in a murine Mϕ cell line (RAW264.7).

Nr4a1 regulates TremL4 expression

The TLR7 ligand R848 activated RAW264.7 cells, resulting in increased CD86, Nr4a1, and TremL4 staining (Figure 10A). lipopolysaccharide (LPS), a TLR4 ligand, had a similar effect (not shown). Nr4a1 mRNA expression increased rapidly after adding R848, peaking at 1 hr (Figure 10B). Treml4 and Tnfa mRNA peaked later on at ~3 hr. TremL4 surface staining also was enhanced by culturing RAW264.7 cells with pristane (12.5–200 ng/ml emulsified in bovine serum albumin [BSA]) (Figure 10C). The effect was dose dependent and specific for pristane, as culture with MO did not enhance TremL4 staining (Figure 10D).

Figure 10

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To further examine Nr4a1 regulation of Treml4 expression, RAW264.7 cells were cultured with LPS in the presence of Nr4a1 or control siRNA. As expected, Nr4a1 siRNA downregulated LPS-stimulated Nr4a1 mRNA expression at 1 hr (Figure 10E). Nr4a1 siRNA also downregulated LPS-stimulated Treml4 mRNA expression at 3 hr. Adherent peritoneal Mϕ from untreated mice behaved similarly. As expected, Nr4a1 expression was minimal in PBS-treated adherent peritoneal Mϕ from Nr4a1−/− mice compared with wild-type controls (Figure 10F, left). Unexpectedly, in adherent peritoneal cells of Nr4a1−/− mice, Nr4a1 increased slightly after LPS stimulation, probably due to the expression of a truncated fragment of the Nr4a1 gene encoding part of the N-terminal domain in B6.129S2-Nr4a1^tm1Jmi/J (Nr4a1−/−) mice (Koenis et al., 2018). TremL4 expression was lower in PBS-treated adherent peritoneal cells from Nr4a1−/− mice vs. wild-type controls (Figure 10F, right). Nr4a1 as well as Treml4 expression was significantly higher in both PBS- and LPS-treated adherent cells from wild-type mice vs. Nr4a1−/− mice (Figure 10F).

Transcriptional profiling of circulating monocytes from uninflamed B6 mice shows a single population of Nr4a1-regulated Ly6C⁻ monocytes (Mildner et al., 2017). These cells mostly remain within the blood vessels where they patrol the vascular endothelium and promote TLR7-dependent repair of damaged endothelial cells (Auffray et al., 2007; Carlin et al., 2013). Previous studies have not fully addressed whether inflammation alters Ly6C⁻ monocytes. We show that Ly6C⁻ monocytes are more heterogeneous during inflammation than in the resting state. Pristane-induced lupus with DAH and small vessel vasculitis is a monocyte/Mϕ-dependent disorder involving lung microvascular endothelial injury (Zhuang et al., 2017; H Zhuang et al., In Revision). Like pristane, MO causes a chronic inflammatory response, but it is not associated with DAH, pulmonary vasculitis, or endothelial damage. We found that Nr4a1-regulated patrolling (Ly6C⁻) monocytes had different phenotypes in mice treated with pristane (CD138⁺) vs. MO (CD138⁻). Pristane directly affected cells of the monocyte/Mϕ lineage, increasing the expression of TremL4, a protein that amplifies TLR7 signaling and altering the normal plasma membrane asymmetry, as shown by increased annexin-V binding to phosphatidylserine on the plasma membrane’s outer leaflet. Ly6C⁻ monocytes from pristane-treated mice produced large amounts of TNFα in response to TLR7 ligand, but despite their high annexin-V staining, did not appear to be more susceptible to apoptosis than Ly6C⁻ monocytes from MO-treated mice. The data suggest that pristane alters the activation state of Ly6C⁻ patrolling monocytes, potentially influencing whether endothelial damage persists or is repaired.

Phenotypes of Ly6C⁻ monocytes in pristane vs. MO-treated mice

Ly6C⁻ monocytes developing from BM-derived Ly6C^hi precursors are thought to be a single-cell population (Sunderkötter et al., 2004; Mildner et al., 2017) that monitors the vascular endothelium and promotes the TLR7-dependent removal of damaged cells (Auffray et al., 2007; Carlin et al., 2013). Development of this lineage requires Nr4a1 (Hanna et al., 2011) and C/EBPβ (Mildner et al., 2017). Circulating monocytes in pristane-treated mice included classical Ly6C^hi inflammatory monocytes (R1) and two subsets of Ly6C⁻ monocytes distinguished by the expression of CD138 and CD11b: Nr4a1-independent CD138⁻CD11b⁺ monocytes (R2/R5) and Nr4a1-regulated CD138⁺CD11b^hi monocytes (R3/R6) (Figure 11). The Nr4a1-independent R2/R5 subset was strongly positive for the monocyte markers CD43 and Treml4 (Figure 2), suggesting that these Ly6C^lo cells are monocytes. Nr4a1-independent Ly6C⁻ monocytes have been encountered before but their function is unclear (Carlin et al., 2013).

Figure 11

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Ly6C⁻CD138⁻ (R2B) Ly6C⁻CD138⁺ (R3B) monocytes were clearly distinguishable by flow cytometry in pristane-treated mice (Figure 5A, C). Consistent with the presence of cells expressing intermediate levels of Ly6C in untreated mice (Mildner et al., 2017), pristane-treated mice also had circulating Ly6C^lo cells that were CD138⁻ (R2A) and CD138⁺ (R3A) (Figure 5A, C). Analogous populations corresponding to R2A/R2B and R3A/R3B were defined by Ly6C and CD11b staining (Figure 5D). The differential requirement for Nr4a1 (Figures 4C and 5B) indicates R2B and R3B are distinct subtypes of Ly6C⁻ monocytes. Consistent with a BM-derived Ly6C^hi precursor (Yona et al., 2013; Sunderkötter et al., 2004; Mildner et al., 2017; Hettinger et al., 2013), R3 monocytes were greatly reduced in pristane-treated Ccr2−/− mice (Figure 3B). In pristane-treated mice, R2 monocytes were unaffected by Ccr2 deficiency and therefore unrelated to R3 monocytes.

Between days 5 and 9 after pristane treatment, circulating R3 monocytes increased significantly while R2 was unchanged (Figure 3A). In MO-treated mice, the opposite pattern was seen: R3 monocytes remained unchanged, whereas R2 increased from days 5 to 9. In pristane-treated Nr4a1−/− mice, the Ly6C^−/loCD138⁺ (R3A/R3B) populations were substantially lower than in wild-type controls, but higher than in PBS- or MO-treated mice (Figure 5B and Figure 11). Due to the low expression of CD138 on monocytes from MO-treated mice, we used an alternative gating strategy to identify subsets of Ly6C^−/lo cells. Ly6C⁻CD11b^hi (R6) and Ly6C⁻CD11b⁺ (R5) monocytes were phenotypically similar to R3 and R2, respectively (Figure 5D and Figure 6). Ly6C⁻ cells (R5B, R6B) in MO-treated mice were Nr4a1 dependent (Figure 9B,C), suggesting that in MO-treated mice, the Nr4a1-dependent Ly6C⁻CD11b^hi subset was CD138−, whereas in pristane-treated mice it was largely CD138+ (Figure 11). Thus, CD138 staining of Ly6C⁻ monocytes was related to the type of inflammatory stimulus (pristane vs. MO).

There are no datasets generated in this current manuscript. The results were put into figures (Figures 1–8).

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Author details

1. Shuhong Han

   Division of Rheumatology, Allergy, & Clinical Immunology, University of Florida, Gainesville, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Supervision, Validation, Methodology, Project administration, Writing - review and editing

   For correspondence

   Shuhong.Han@medicine.ufl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6951-120X

2. Haoyang Zhuang

   Division of Rheumatology, Allergy, & Clinical Immunology, University of Florida, Gainesville, United States

   Contribution

   Conceptualization, Data curation

   Competing interests

   No competing interests declared

3. Rawad Daniel Arja

   Division of Rheumatology, Allergy, & Clinical Immunology, University of Florida, Gainesville, United States

   Contribution

   Data curation, Software

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4555-2098

4. Westley H Reeves

   Division of Rheumatology, Allergy, & Clinical Immunology, University of Florida, Gainesville, United States

   Contribution

   Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing - original draft, Project administration, Writing - review and editing

   For correspondence

   westley.reeves@medicine.ufl.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2924-4549

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