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. Author manuscript; available in PMC: 2016 Jun 16.
Published in final edited form as: Immunity. 2015 Jun 9;42(6):1130–1142. doi: 10.1016/j.immuni.2015.05.011

Bone marrow-resident NK cells prime monocytes for regulatory function during infection

Michael H Askenase 1,2, Seong-Ji Han 1, Allyson L Byrd 1,3, Denise Morais da Fonseca 1, Nicolas Bouladoux 1, Christoph Wilhelm 1, Joanne E Konkel 4,5, Timothy W Hand 1, Norinne Lacerda-Queiroz 6, Xin-Zhuan Su 6, Giorgio Trinchieri 7, John R Grainger 1,4,5,*, Yasmine Belkaid 1,*
PMCID: PMC4472558  NIHMSID: NIHMS693740  PMID: 26070484

SUMMARY

Tissue-infiltrating Ly6Chi monocytes play diverse roles in immunity, ranging from pathogen killing to immune regulation. How and where this diversity of function is imposed remains poorly understood. Here we show that during acute gastrointestinal infection, priming of monocytes for regulatory function preceded systemic inflammation and was initiated prior to bone marrow egress. Notably, natural killer (NK) cell-derived IFN-γ promoted a regulatory program in monocyte progenitors during development. Early bone marrow NK cell activation was controlled by systemic interleukin-12 (IL-12) produced by Batf3-dependent dendritic cells (DC) in the mucosal-associated lymphoid tissue (MALT). This work challenges the paradigm that monocyte function is dominantly imposed by local signals following tissue recruitment, and instead proposes a sequential model of differentiation in which monocytes are pre-emptively educated during development in the bone marrow to promote their tissue-specific function.

INTRODUCTION

Tissue inflammation induces the rapid recruitment of circulating myeloid cells that can both generate inflammatory responses that protect against infection as well as mediate regulatory responses and tissue repair. Ly6Chi monocytes in particular can perform multiple roles upon tissue recruitment, and the differentiation and effector function of these cells can be shaped by the local tissue environment, both at steady-state and during inflammation (Bain et al., 2013; Tamoutounour et al., 2013; Zigmond et al., 2012), For instance, local acquisition of regulatory mediators, such as interleukin-10 (IL-10), by recruited monocytes has been described in the healthy gut and inflamed skin, as well as during muscle repair (Arnold et al., 2007; Bain et al., 2013; Egawa et al., 2013), and Ly6Chi monocytes can give rise to immunosuppressive tumor-associated macrophages in cancer (Franklin et al., 2014). From these studies, a paradigm has emerged proposing that local tissue signals are largely responsible for the acquisition of appropriate function and fate of Ly6Chi monocytes following tissue entry (Bain and Mowat, 2014; Ingersoll et al., 2011; Serbina et al., 2008).

Increased bone marrow (BM) output of inflammatory cells, known as “emergency myelopoiesis”, is a critical feature of the host response to injury or infection. This process can be driven by systemic inflammatory factors and/or pathogen-derived products acting on precursor cells (Takizawa et al., 2012). Long-term changes to monocyte function favoring microbicidal potential following infection have been recently documented in a process termed “trained immunity” (Cheng et al., 2014; Quintin et al., 2012). It remains unknown, however, if in defined inflammatory settings, discrete signals could alter monocyte function during development prior to the onset of systemic inflammation.

Early education of monocytes may be particularly important for controlling immune responses at highly reactive sites such as the gut. In this context, we previously identified a dual role for Ly6Chi monocytes during acute intestinal infection with Toxoplasma gondii in limiting responses to commensal microbes and preventing lethal immunopathology (Grainger et al., 2013). Here, we used this model to examine how and where monocytes become primed for regulatory functions. We found that, contrary to the prevailing paradigm, the capacity of Ly6Chi monocytes to respond to microbial stimuli can be altered during their development and prior to BM egress. These results reveal that the functional diversity of monocytes may be imprinted early in development and uncover a role for BM innate lymphoid cells in altering haematopoiesis in order to generate effector cells optimally programmed to control tissue-specific immunity.

RESULTS

Systemic alteration of Ly6Chi monocytes prior to tissue recruitment

Following per-oral infection with T. gondii, Ly6Chi monocytes play an important role in protecting against the pathogen (Dunay et al., 2008), as well as regulating pathologic responses towards the microbiota via prostaglandin E2 (PGE2) production (Grainger et al., 2013). We assessed whether the potential for regulatory function was imposed by signals in the mucosal environment or acquired prior to tissue recruitment. To this end, we first explored the possibility that functional alterations to monocytes were coupled with a distinct surface phenotype. As previously described (Grainger et al., 2013), all Ly6Chi monocytes from T. gondii-infected small intestine lamina propria (SILP) expressed MHCII (Figure 1A, S1A). Moreover, in contrast to their naïve counterparts, Ly6Chi monocytes from infected mice expressed high levels of stem cell antigen 1 (Sca-1) (Figure 1A). However, unlike monocytes described in other acute inflammatory settings (Tamoutounour et al., 2013; Zigmond et al., 2012), Ly6Chi monocytes did not express fractalkine receptor (CX3CR1) (Figure 1A). Thus, monocyte regulatory properties in the gut are associated with a previously un-described phenotypic signature, an observation that provided us with the opportunity to track when and where monocyte regulatory priming occurred.

Figure 1. Monocytes are educated prior to tissue entry during infection.

Figure 1

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CX3CR1-GFP mice were infected per-orally with T. gondii. (A) Flow-cytometric analysis of Ly6Chi monocytes (Mo) present in the small intestinal lamina propria (SILP) of naïve animals or at day 8 post-infection (p.i.). (B) Phenotype of blood Ly6Chi monocytes at defined time-points p.i. as measured by flow cytometry. (C) Frequency of blood Ly6Chi monocytes within the total blood monocyte compartment. (D) Ex vivo PGE2 production by Ly6Chi monocytes sorted from the blood of naïve or day 8 infected mice and cultured in the presence or absence of E. coli lysate. (E) IL-10 and TNF-α production by blood Ly6Chi monocytes from naïve and day 8 T. gondii infected animals cultured in the presence or absence of E. coli lysate. (F) Accumulation of monocytes in the SILP. (G) Serum levels of IFN-γ and TNF-α in infected mice. Dots represent individual animals. Error bars represent standard deviation. Data are representative of at least two independent experiments, n = 3-6 per group. Statistical comparisons were performed using one-way ANOVA or unpaired student's t test adjusted for multiple comparisons. **: p<0.01, ***: p<0.001. See also Figure S1.

This phenotype was detectable in the blood as early as day 4 post-infection and adopted by all Ly6Chi monocytes by day 6 (Figure 1B, S1B, S1C). In addition to Ly6Chi monocytes, the blood is home to a population of patrolling Ly6Clo monocytes important for repair and maintenance of the endothelium (Carlin et al., 2013). Concurrent with changes to their phenotype, the Ly6Chi monocyte subset rapidly became the dominant monocyte population present in the circulation during infection (Figure 1C).

As in the gut, these phenotypic changes in the blood compartment were associated with the acquisition of regulatory responses. The potential to express IL-10 was increased in circulating Ly6Chi monocytes following infection and Ly6Chi monocytes isolated from the blood of infected mice produced greater quantities of PGE2 and IL-10 upon stimulation with E. coli lysate (Figure 1D, 1E, S1D). E. coli lysate represents a relevant source of stimulation for monocytes homing to the gut, since during acute mucosal infections the gut becomes dominated by γ-proteobacteria such as E. coli that contribute to inflammation (Heimesaat et al., 2006; Molloy et al., 2013). In contrast to granulocyte colony stimulating factor (G-CSF)-induced regulatory monocytes (D'Aveni et al., 2015), acquisition of anti-inflammatory potential by monocytes following infection was not associated with expression of CD34 (Figure S1E). As in the gut, monocytes in the blood maintained their effector potential upon acquisition of regulatory function, producing TNF-α upon stimulation (Figure 1E).

Surprisingly, the onset of systemic alterations to Ly6Chi monocytes preceded monocyte recruitment to the SILP, increases in systemic IFN-γ or TNF-α, and detectable intestinal pathology (Figure 1F, 1G, S1F, S1G, S1H). Additionally, these changes were not the consequence of parasite dissemination from the MALT (Figure S1I). Thus, following infection, early phenotypic alterations to monocytes are associated with profound changes to their function in both the target tissue and in the blood compartment.

IFN-γ remodels the blood monocyte compartment during infection

We next assessed whether acquisition of the MHCII+Sca-1+CX3CR1− phenotype by monocytes was a common response to infection. To this end, mice were infected with Yersinia pseudotuberculosis and Plasmodium yoelii, pathogens known to induce type 1 immune responses (Doolan and Hoffman, 1999; Logsdon and Mecsas, 2006). In both settings, Ly6Chi monocytes acquired a similar phenotype to that observed during T. gondii infection and became the dominant blood monocyte subset (Figure 2A, 2B). This observation supported the idea that monocyte regulatory priming was likely driven by a canonical mediator of host defense employed during these responses, rather than by interaction with a specific pathogen. Indeed, following injection of recombinant IFN-γ for three days, blood Ly6Chi monocytes ubiquitously expressed Sca-1 and MHCII and demonstrated decreased expression of CX3CR1 (Figure 2C). Moreover, IFN-γ increased the proportion of circulating Ly6Chi monocytes (Figure 2D). Conversely, blockade of IFN-γ during T. gondii infection prevented these phenotypic and subset alterations (Figure 2E, 2F).

Figure 2. IFN-γ remodels the blood monocyte compartment.

Figure 2

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(A-B) CX3CR1-GFP mice were infected intravenously with P. yoelii infected red blood cells, per-orally with Y. pseudotuberculosis, or per-orally with T. gondii. (A) Phenotype of blood Ly6Chi monocytes (Mo) following infection with P. yoelli (day 4 p.i.), Y. pseudotuberculosis (day 5 p.i.) and T. gondii (day 8 p.i.). (B) Frequency of blood Ly6Chi monocytes within the total monocyte compartment at time-points described in (A). (C-D) CX3CR1-GFP mice were administered IFN-γ or PBS once per day for three consecutive days. (C) Phenotype of blood Ly6Chi monocytes assessed by flow cytometry. (D) Frequency of Ly6Chi blood monocytes within the total monocyte compartment. (E-F) CX3CR1-GFP mice were infected with T. gondii and treated with anti-IFN-γ Ab or isotype control (IgG). (E) Phenotype of blood Ly6Chi monocytes. (F) Frequency of Ly6Chi monocytes within the total blood monocyte compartment. (G-H) Chimeric mice comprised of equal numbers of WT CD45.1+ and Ifngr1−/− (CD45.2+) leukocytes were infected or not with T. gondii. (G) Mean fluorescent intensity (MFI) of MHCII and Sca-1 expression by WT and Ifngr1−/− blood Ly6Chi monocytes measured by flow cytometry at 8 days p.i. Values of WT and Ifngr1−/− cells from the same host are joined by a line. (H) Proportion of blood Ly6Chi monocytes in total blood monocytes within WT and Ifngr1−/− compartments in naïve and infected mice. Error bars represent standard deviation. Data are representative of at least two independent experiments, n = 3-5 per group. Statistical comparisons were performed using unpaired student's t test, or paired t test adjusted for multiple comparisons. **: p<0.01, ***: p<0.001.

We next addressed whether this phenomenon was the consequence of cell intrinsic or extrinsic responses to IFN-γ. To this end, we generated mixed BM chimeras with WT cells and cells lacking IFN-γ receptor (Ifngr1−/−) that were subsequently infected with T. gondii. Changes to monocyte surface phenotype as well as alterations in proportion of Ly6Chi monocytes were dependent on cell intrinsic IFN-γ receptor signaling (Figure 2G, 2H).

Ly6Chi monocytes are functionally primed in the bone marrow

Our results thus far supported the idea that monocytes can be primed for regulatory function prior to BM egress. To explore this possibility, Ly6Chi monocytes were isolated from BM at day 5 post-infection and stimulated ex vivo with various bacterial or parasite-derived ligands. Even at this early time-point, BM monocytes had already acquired enhanced capacity to produce PGE2 in response to several bacterial ligands, but not parasite ligands, compared to cells isolated from naïve mice (Figure 3A). Enhanced PGE2 production by BM monocytes was still detectable at 24 days post-infection (Figure 3B), past the acute phase of disease (Grainger et al., 2013; Molloy et al., 2013).

Figure 3. IFN-γ primes BM monocytes for regulatory function during infection.

Figure 3

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(A) Ex vivo PGE2 production by Ly6Chi monocytes sorted from BM of naïve or T. gondii infected mice at day 5 p.i. and cultured with or without E. coli lysate, Soluble Toxoplsama antigen (STAg), or various TLR ligands. (B) Ex vivo PGE2 production by BM Ly6Chi monocytes sorted from naïve or infected mice at day 24 p.i. and stimulated with E. coli lysate. (C) WT mice were infected as in (A) and treated with anti-IFN-γ Ab or isotype control. Ly6Chi monocytes were sorted from BM of naïve and infected mice and assessed ex vivo for PGE2 production in response to E. coli lysate. (D) Ex vivo PGE2 production by Ly6Chi monocytes sorted from BM of CX3CR1-GFP mice treated with IFN-γ or PBS once per day for three consecutive days. (E) Ex vivo PGE2 production by BM Ly6Chi monocytes sorted from naïve WT mice, cultured for 6 hours with or without IFN-γ, and subsequently stimulated with E. coli lysate. (F-G) WT mice were infected as in (A) and BM Ly6Chi monocytes were sorted from naïve or day 5 infected mice. Sorted monocytes were cultured for 6 hours in the presence or absence of LPS, and gene expression assessed using the NanoString platform. (F) Principle component analysis comparing gene expression of untreated and LPS stimulated monocytes from naïve and infected mice. Plot represents clustering of samples in a 2-dimensional matrix of principle components 1 and 2. (G) Gene expression of untreated naïve monocytes from (C) was compared to each of the other three groups. Numbers in each section of the Venn diagram represent the number of genes altered in expression between that group and untreated naïve controls, with overlapping regions representing genes changed in more than one group. (H) Heat maps representing the relative expression of the 30 genes changed only in monocytes from infected mice upon LPS stimulation. Error bars represent standard deviation. Data are representative of two or more independent experiments, n = 3-5 replicates per group (A-E). Columns represent biological replicates of 2 pooled samples (H). Statistical comparisons were performed using unpaired student's t test, adjusted for multiple comparisons (A-E) or by Welch's t test adjusted for multiple comparisons (F-H). *: p<0.05, **: p<0.01, ***: p<0.001.

Acquisition of this regulatory function by BM monocytes was dependent on signaling by IFN-γ, as in vivo blockade of IFN-γ during infection returned PGE2 production to baseline levels (Figure 3C). Furthermore, administration of IFN-γ to naïve mice was sufficient to drive increased PGE2 production by BM monocytes, and ex vivo IFN-γ treatment of monocytes isolated from naïve mice enhanced PGE2 production (Figure 3D, 3E).

To more comprehensively explore the early consequences of infection on the function of monocytes, sorted BM Ly6Chi monocytes from naïve or d5 T. gondii infected animals were cultured in the presence or absence of LPS, and mRNA expression of 490 myeloid genes was assessed using the NanoString platform. Principle component analysis revealed that monocytes from T. gondii infected animals demonstrated a transcriptional program distinct from those isolated from naïve controls, when cultured in media alone as well as upon LPS stimulation (Figure 3F, Table S1).

We next compared the gene expression of untreated BM Ly6Chi monocytes from naïve animals to that of each of the other groups (naïve + LPS stimulated, T. gondii infected untreated, T. gondii infected + LPS stimulated) (Figure 3G). Our analysis revealed that in untreated monocytes from infected animals, a greater than 2-fold change in expression of 33 genes was observed (Figure 3G, purple circle). Moreover, following infection, the responsiveness of BM monocytes to LPS stimulation was dramatically altered. In total, 88 common genes were differentially expressed upon LPS stimulation in monocytes from both naïve and infected animals, but an additional 30 genes were altered in expression only in monocytes from infected animals (Figure 3G, 3H).

In support of our functional read out, expression of Ptgs2, the gene that encodes COX-2, a key enzyme controlling PGE2 production during inflammation (Ricciotti and FitzGerald, 2011), was induced in response to LPS stimulation in monocytes from infected mice (Figure 3H). Further supporting the idea that monocytes were primed for increased regulatory function prior to BM egress, gene expression of Cd200, which encodes a surface protein that suppresses innate immune cell function (Snelgrove et al., 2008), and Socs3, which encodes a negative regulator of inflammatory cytokine signaling (Carow and Rottenberg, 2014), was also increased. Monocytes from infected mice also augmented the expression of genes encoding pro-inflammatory factors upon LPS stimulation, including IL-1β and IL-12, suggesting a generally increased responsiveness to bacterial ligands. These results imply that early during infection and prior to BM egress, monocytes are primed for both regulatory and effector responses following exposure to bacterial stimuli.

IFN-γ controls the transcriptional program of monocyte progenitors

Monocytes are constitutively generated in the BM, developing through a series of well-defined stages including the granulocyte monocyte precursor (GMP), monocyte dendritic cell precursor (MDP), and the more recently described common monocyte progenitor (cMoP) (Fogg et al., 2006; Hettinger et al., 2013). Notably, we found that early during the infection, cMoP and BM monocytes began to acquire the distinct phenotype associated with acquisition of regulatory function (Figure 4A, S2A, S2B). These phenotypic changes persisted following resolution of the acute phase of the infection (Figure S2C-E).

Figure 4. IFN-γ controls gene expression of monocyte progenitors early during infection.

Figure 4

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(A) CX3CR1-GFP mice were infected with T. gondii. At defined time-points p.i., the phenotype of monocyte progenitors (cMoP) present in the BM was assessed by flow cytometry. (B) cMoP were sorted from the BM of naïve or day 5 infected mice, and gene expression was assessed by NanoString. Heat maps represent the relative expression of 37 genes by cMoP selected by pathway analysis as being affected by IFN-γ signalling from naïve and infected mice. (C) CX3CR1-GFP mice were administered 5 μg IFN-γ or PBS by i.p. injection once per day for three consecutive days. Mice were sacrificed eighteen hours after the final injection, and MHCII and Sca-1 expression by cMoP was assessed. (D) CX3CR1-GFP mice were infected and administered anti-IFN-γ Ab or isotype control. Expression of MHCII and Sca-1 was assessed at day 5 p.i. Error bars represent standard deviation. Data are representative of two or more independent experiments, n = 3-5 mice per group (A, C, D). Columns represent individual samples pooled from 3 mice (B). Statistical comparisons were performed using one-way ANOVA (A, D), Welch's t test adjusted for multiple comparisons (B), or unpaired students t test (C). ***: p<0.001. See also Figures S2, S3, and S4.

We next assessed how mucosal infection changed the transcriptional response of monocyte progenitors in the BM. At day 5 post infection, of the 490 genes assessed, 91 genes had a greater than 2-fold change (FDR <0.05) in expression in the cMoP from naïve versus T. gondii infected animals (Table S1). Pathway analysis revealed a distinct interferon signature, marked by increased expression of 8 transcriptional regulators, including Stat1, Stat2, and Irf7 (Figure 4B). Furthermore, a broad suite of anti-microbial factors were increased in expression, including Gbp2 and Ifi47, intracellular defense factors critical for resistance to T. gondii infection (Collazo et al., 2001; Degrandi et al., 2013). In agreement with our flow cytometric characterization, expression of transcripts for MHCII (H2-Aa, H2-Ab1, H2-Eb1) and Sca-1 (Ly6a) were increased during infection, while expression of Cx3cr1 was decreased. These results reveal that the transcriptional program of monocyte progenitors during infection can be dramatically altered in the BM prior to terminal differentiation and egress.

Despite these transcriptional changes, cMoP remained committed to the monocyte lineage during infection as evidenced by the sustained expression of Irf8, a transcription factor critical for monocyte development (Kurotaki et al., 2013) (Figure S3A), and lack of expression of transcripts associated with the DC lineage such as Zbtb46, Flt3, and Id2 (Merad et al., 2008) (Figure S3B). Further, when cultured under monocyte differentiation conditions (Hettinger et al., 2013), MHCII+Sca-1+ putative cMoP from infected mice differentiated into Ly6ChiCD11bhicKit− mature monocytes (Figure S3C, S3D). Likewise, when cultured under conditions favoring macrophage differentiation (Hettinger et al., 2013), MHCII+Sca-1+ putative monocytes differentiated into Ly6CloF4/80hi macrophages (Figure S3E, S3F). These macrophages differentiated from MHCII+Sca-1+ monocytes lacked expression of MHCII or Sca-1, making them phenotypically identical to those derived from naïve animals (Figure S3G). This suggested that expression of MHCII and Sca-1 required sustained IFN-γ signalling.

Indeed, administration of IFN-γ to naïve mice was sufficient to increase expression of MHCII and Sca-1 and decrease expression of CX3CR1 (Figure 4C, Figure S4A, S4B). Acquisition of this phenotype during T. gondii infection was impaired in cells lacking a functional IFN-γ receptor (Figure S4C, S4D) and following IFN-γ blockade (Figure S4E, S4F). Additionally, treatment with anti-IFN-γ abolished the transcriptional program adopted by cMoP during infection (Figure S4G). Thus, IFN-γ can directly shape the transcriptional program and function of monocytes during their development.

NK cell production of IFN-γ in the BM educates monocyte progenitors

The extent of cMoP and monocyte education as early as day 5 post-infection and prior to systemic inflammation raised the possibility that these changes may be triggered by IFN-γ production in the bone marrow. Indeed, phenotypic alterations to BM monocytes during T. gondii infection preceded significant increases in serum levels of IFN-γ (Figure 1G, S2B). The BM is home to mature immune cells capable of producing IFN-γ, such as T cells and innate lymphoid cells (ILC), including classical NK cells (Becker et al., 2005; Klose et al., 2014). To identify the early source of IFN-γ in the BM during infection, we tracked the production of this cytokine by flow cytometry using direct ex vivo staining and IFN-γ reporter mice (Reinhardt et al., 2009), as well as by imaging. We found that BM-resident NK cells produce IFN-γ as early as day 4 post-infection (Figure 5A, 5B, S5A). At this time-point, NK cells were the dominant source of IFN-γ in the BM, with minimal contribution by T cells or type 1 ILC, while at day 5, IFN-γ production was also observed in T cells (Figure 5B, S5B, S5C). Histological analysis at day 5 post-infection found IFN-γ producing NK cells in close proximity to BM monocytes (Figure 5C, 5D, S5D).

Figure 5. BM NK cells educate cMoP and Ly6Chi monocytes via IFN-γ.

Figure 5

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(A) WT mice were infected with T. gondii. At defined time-points, lymphocyte IFN-γ production was assessed by flow cytometry. Plots are gated on TCR-β−NK1.1+DX5+ NK cells. (B) Absolute numbers of lymphocytes producing IFN-γ in the BM. (C-D) Confocal images of BM of IFN-γ YFP mice at day 5 p.i. (C) Yellow arrows indicate NK cells producing IFN-γ. (D) Ly6G−Ly6B2+ monocytes in contact with (yellow arrows) or in close proximity to (orange arrows) NK cells producing IFN-γ. (E) CX3CR1-GFP mice were infected as in (A) and injected intravenously with NK cell depleting antibody (αNK1.1) or isotype control. MHCII and Sca-1 expression by cMoP were assessed at day 5 p.i. Error bars represent standard deviation. Data are representative of two or more independent experiments, n = 3-5 mice per group. Statistical comparisons were performed using oneway ANOVA. **: p<0.01, ***: p<0.001. See also Figure S5.

To further assess whether NK cell-derived IFN-γ could directly impact cMoP, we treated mice with anti-NK1.1 depleting antibody. This treatment resulted in nearly complete ablation of the NK compartment in most tissues, while NK cells in the BM were decreased by approximately 75% (Figure S5E). NK depletion during infection significantly inhibited the early acquisition of Sca-1 and MHCII by BM cMoP and Ly6Chi monocytes, and reversed changes to CX3CR1 expression (Figure 5E, S5F, S5G). NK depletion also reduced the changes to monocyte phenotype in the blood, although the effects were less pronounced than on BM populations, suggesting that other sources of IFN-γ may further educate monocytes outside of the BM (Figure S5H).

IL-12 produced by Batf3-dependent DC in the MALT induces IFN-γ production by BM NK cells

T. gondii is a potent inducer of IL-12, a cytokine involved in NK cell activation (Trinchieri, 2003). Following infection, a rapid increase in IL-12p70 was detectable in the blood that coincided with NK activation in the BM (Figure 6A). Blockade of IL-12 prevented IFN-γ production by BM-resident NK cells and impaired MHCII and Sca-1 expression by cMoP and monocytes (Figure 6B, 6C, S6A, S6B). Early alterations in cMoP and monocytes were likewise abolished in IL-12p35 deficient animals, but not in mice deficient in IL-18, another potent stimulator of IFN-γ production from NK cells (Scharton-Kersten et al., 1996) (Figure S6C-E). Moreover, administration of recombinant IL-12 alone was sufficient to drive IFN-γ production by NK cells and phenotypic alterations to cMoP and monocytes (Figure 6D, 6E, S6F, S6G).

Figure 6. IL-12 produced by Batf3-dependent DC in the MALT stimulates NK cells to secrete IFN-γ.

Figure 6

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(A) Serum IL-12p70 levels post-infection. (B-C) WT mice were infected with T. gondii and treated with anti-IL-12p70 Ab or isotype control. (B) Absolute numbers of IFN-γ producing lymphocytes at day 5 p.i. (C) MHCII and Sca-1 expression by cMoP at day 5 p.i. (D-E) WT mice were administered IL-12 or PBS once per day for two days. (D) Absolute numbers of IFN-γ producing lymphocytes in the BM. (E) MHCII and Sca-1 expression by cMoP. (F) Batf3−/− mice and WT controls were infected and CD8α+ and CD11b+ resident DC in the mesLN were assessed at day 5 p.i. Dot plots are gated on TCR-β−CD11c+MHCIIint resident DC. Numbers represent mean frequency of cells gated in this manner +/− standard deviation. (G) Serum IL-12p70 in Batf3−/− and WT mice, naïve and day 5 p.i.. (H) Absolute numbers of IFN-γ producing lymphocytes in Batf3−/− mice and WT controls at day 5 p.i. (I) MHCII expression by cMoP in Batf3−/− mice and WT controls at day 5 p.i. (J) Ten percent of the total cells from each tissue were cultured for 16 hours and cell supernatants were assessed for IL-12p70. Error bars represent one standard deviation. Data are representative of two or more independent experiments (A-I) or the pooled results of 2 independent experiments (J), n = 3-5 per group. Statistical comparisons were performed using one-way ANOVA or unpaired student's t test, corrected for multiple comparisons. *: p < 0.05, **: p<0.01, ***: p<0.001. See also Figure S6.

We next assessed the tissue and cellular source of IL-12 during T. gondii infection. Previous work revealed a dominant role for Batf3-dependent DC in IL-12 production during intra-peritoneal T. gondii infection (Mashayekhi et al., 2011). Consistent with this, in Batf3 deficient mice that are devoid of CD8α+ lymphoid resident DC and migratory CD103+CD11b− DC (Edelson et al., 2010; Hildner et al., 2008), IL-12p70 was not increased in the serum upon oral infection and early NK cell activation was not detected (Figure 6F-H, S6H). Furthermore, reduced NK activation in these mice was associated with impaired acquisition of the Sca-1+MHCII+ phenotype by BM cMoP and monocytes (Figure 6I, S6I-K).

Notably, in contrast to the spleen and BM, the mesLN compartment produced detectable levels of IL-12p70 post-infection (Figure 6J). Additionally, transcriptional analysis of Batf3-dependent DC in the mesLN and spleen revealed a significant increase in Il12b expression in mesLN DC but not splenic DC post-infection (Figure S6L). Although we cannot completely exclude the possibility of a transient and discrete contribution of the BM, our present results, in agreement with previous studies (Oldenhove et al., 2009; Washino et al., 2012), support the idea that the MALT is the dominant source of early IL-12 following per-oral T. gondii infection.

Thus, we propose a model in which mucosal infection can result in early IL-12 production in the MALT, which acts in a systemic manner to activate NK cells in the BM, resulting in IFN-γ production in this primary hematopoietic tissue. Although we cannot dismiss a potential role of systemic IFN-γ later in this process, our findings suggest that early IFN-γ-producing NK cells present in the BM can act as an early alarm system to rapidly prime Ly6Chi monocytes to respond in a regulatory manner upon their arrival at barrier sites. Collectively, these data describe an unexpected role for the local production of IFN-γ in the BM during mucosal infection in altering monocyte function and reveal that monocyte functional education is a sequential process in which discrete signals act in defined spatially and temporally segregated niches.

DISCUSSION

It is often assumed that the tissue microenvironment is largely responsible for determining the function of recruited cells (Bain and Mowat, 2014; Ingersoll et al., 2011; Serbina et al., 2008). Our present work proposes a pre-emptive model in which discrete signals received during development can also have profound consequences on the subsequent function of myeloid cells in defined tissue environments. To our knowledge, we describe here for the first time transcriptional reprogramming of myeloid progenitors in response to inflammatory factors produced locally in the BM prior to the onset of a systemic inflammatory response. Our results support the idea that early sensing of IFN-γ (Signal 1) by cMoP in the BM educates the subsequent function of Ly6Chi monocytes, particularly their capacity to generate regulatory responses toward products derived from the microbiota (Signal 2). Together, we believe that our present work uncovers a general mechanism of regulation designed to prepare monocytes for recruitment to barrier tissues and prevent aberrant reactivity to the microbiota that can lead to heightened immunopathology and tissue damage.

A number of recent studies have noted that upon infection, NK cells can rapidly become activated and form clusters in close association with Ly6Chi monocytes, priming these cells via IFN-γ production and thus enhancing their anti-microbial function (Coombes et al., 2012; Goldszmid et al., 2012; Kang et al., 2008). Notably, in each of these cases, the activation of NK cells was proposed to occur in response to local IL-12 signaling. Here we show that this activation process can occur in the hematopoietic environment in response to IL-12 emanating from a distally infected tissue. In this setting, IFN-γ-producing NK cells educate monocytes and their progenitors prior to BM egress and pathogen encounter. Our findings suggest that a previously unappreciated function of mature NK cells present in the BM is to act as a local alarm system following infection of barrier sites, shaping hematopoiesis to favor production of optimally educated effector cells.

Previously, Batf3-dependent DC have been implicated in the development of protective immunity towards T. gondii during intraperitoneal infection, primarily due to their capacity to make IL-12 in response to T. gondii-derived ligands (Mashayekhi et al., 2011). Here, we show that a brief burst of IL-12 production by this population in the MALT early during infection can activate NK cells in the BM. These data raise the possibility that one of the roles of Batf3-dependent DC in the MALT is to signal from the barrier to the BM during infection, thereby initiating the generation of a monocyte primed for regulatory function. Thus, Batf3-dependent DC may not only promote the induction of adaptive Th1 immunity, but also control the fate and function of inflammatory cells during their development via their capacity to distally control hematopoiesis prior to systemic inflammation.

Acute primary infection can epigenetically reprogram monocytes, enhancing effector function against subsequent heterologous infection (Cheng et al., 2014; Quintin et al., 2012), a phenomenon known as “trained immunity”. Intriguingly, we found that not only did early signals during T. gondii infection modulate the phenotype and function of monocytes during infection, but that these changes persisted after resolution of acute inflammation. In this particular setting of infection, T. gondii establishes a low-grade chronic infection, therefore, whether the long-term effects we observe are dependent on the initial BM priming or low levels of systemic IFN-γ remains unclear.

Overall, our findings present a new paradigm for how monocyte fate and function are educated during infection. The critical roles of IL-12, IFN-γ, and bacterial stimuli in classical activation of macrophages and their anti-microbial function have been studied for decades, but how the appropriate sequence of signals leads to coordinated tissue responses has remained unclear. In particular, our study highlights the highly ordered temporal nature of these signals and the discrete compartments in which each of these factors acts. This procession of signals ultimately results in a balance of inflammatory and regulatory function by Ly6Chi monocytes upon recruitment to the inflamed gastrointestinal tract. Further research into how monocyte development and function are educated to mediate this balance may provide insight into the role these cells play in perpetuating inflammation in acute and chronic disease. Moreover, better understanding of the mechanisms controlling monocyte regulatory function, and particularly the importance of BM lymphocytes in early control of this process, may lead to the identification of novel therapeutic modalities in settings where aberrant monocyte activity has been implicated, such as in inflammatory bowel diseases (IBD) and cancer.

EXPERIMENTAL PROCEDURES

Mice

All mice were bred and maintained under pathogen-free conditions at an American Association for the Accreditation of Laboratory Animal Care-accredited animal facility at the NIAID and housed in accordance with the procedures outlined in the Guide for the Care and Use of Laboratory Animals. All experiments were performed under an animal study proposal approved by the NIAID Animal Care and Use Committee. Gender- and age-matched mice between 6-14 weeks of age were used. Refer to the Supplemental Experimental Procedures for more information on specific mouse strains.

T. gondii Parasite and Infection Protocol

ME-49 clone C1 of T. gondii (provided by Dr. Michael Grigg, NIAID/NIH) was obtained by electroporation of the parental ME-49 type II strain (ATCC 50840) with red fluorescent protein (RFP) and was used for production of tissue cysts in C57BL/6 mice. Tissue cysts used in experiments were obtained from female mice that were per-orally inoculated with 10 cysts 2-3 months earlier. Animals were euthanized and their brains removed and homogenized in 1 ml of PBS pH 7.2. Cysts were counted on a fluorescent microscope. For experiments, mice were infected by intragastric gavage with 10 cysts of ME-49 C1.

Ly6Chi Monocyte and cMoP Purification by FACS

Cell suspensions of naïve or T. gondii infected SILP, blood, or BM were incubated with mixtures of monoclonal antibodies containing anti-FcγIII/II, mouse serum, Rat IgG (Jackson Immunoresearch), and 7-AAD viability staining solution (eBioscience) for 15 minutes on ice. After staining, cell suspensions were washed, filtered, resuspended in complete medium without phenol red and sorted on a BD FACSAria II.

Ex vivo Stimulation of FACS purified Ly6Chi Monocytes for PGE2 Detection

Purified Ly6Chi monocytes were cultured in complete RPMI at a concentration of 150,000 cells/mL (for monocytes derived from BM) or 30,000 cells/mL (for monocytes derived from blood) in a 96-well round-bottom tissue culture plate. In some cases, cells were stimulated with E. coli lysate (1 μg/ml), Soluble Toxoplasma antigen (STAg) (1 μg/ml), or commercially available TLR ligands Pam2CSK4 (Invivogen, 500 nM), LPS (Enzo Life Sciences, 500 ng/ml), Flagellin (Invivogen, 500 ng/ml), CpG (5’TCCATGACGTTCTGAT3’), Integrated DNA Technologies, 500 nM), or Profilin (Alexis Biochemicals, 500 ng/ml) for 18 hours. STAg was prepared as previously described (Grunvald et al., 1996). Three to five replicates of pooled supernatants from 4-5 mice were assayed for PGE2 using an enzyme-immunoassay (EIA) (Cayman Chemicals), as per manufacturers’ instructions. In some experiments, cells were incubated with 20 ng/ml recombinant IFN-γ (BioLegend) for 6 hours and washed prior to stimulation with E. coli lysate.

Ex vivo Stimulation of FAC-Purified Ly6Chi Monocytes for NanoString Analysis

Ly6Chi monocytes FACS sorted from the BM of uninfected mice or T. gondii infected mice at day 5 post-infection were cultured for 6 hours in complete RPMI at a concentration of 30,000 cells in 100 mL of complete media in a 96 well v-bottom plate. In some cases, cells were stimulated with 500 ng/ml LPS (Enzo Life Sciences). After 6 hours, cells were washed and resuspended at 2,000 cells/μL of RLT buffer (Life Technologies) for RNA isolation and stored at −80° C.

Gene Expression Analysis by NanoString nCounter Technology

The nCounter analysis system (NanoString Technologies) was used to assess gene expression by, cMoP, BM Ly6Chi monocytes, and various lymphoid resident DC populations. Briefly, RNA was obtained by lysing the sorted cells (5-10 × 103 cells/5 μL) in RLT buffer (Qiagen) and then hybridized with the C2566 Mouse Myeloid Panel. Data analysis was performed according to NanoString Technology recommendations. For more information, please refer to the Supplemental Experimental Methods.

Intracellular Detection of IFN-γ

For ex vivo IFN-γ detection, cell suspensions from BM were cultured at 5 × 105 - 1 × 106 cells/well in 96-well round-bottom plates in the presence of 1μg/mL brefeldin A (GolgiPlug, BD Biosciences). After 3 hours, cells were stained for surface markers, then washed twice with FACS buffer and fixed in a solution of 2% paraformaldehyde (Electron Microscopy Sciences). Prior to fixation, Live/Dead Fixable Blue Cell Stain Kit (Invitrogen) was used to exclude dead cells. Cells were then stained with PE-conjugated antibody against IFN-γ (XMG1.2, eBioscience) or isotype control in the presence of anti-FcεIII/II receptor for 60 minutes on ice in FACS buffer containing 0.5% saponin.

Immunofluorescence imaging

For immunofluorescence staining of whole-mounted tissues and frozen sections of BM, we adapted a previously published protocol (Kunisaki et al., 2013). Femoral bones were perfused with Periodate-lysine-paraformaldehyde (PLP) fixation buffer. Post perfusion femurs were fixed for at least 10 hours in PLP-buffer at 4°C, incubated in 30% sucrose overnight, embedded in optical cutting temperature compound (OCT) (Sakura Finetek) over dry ice and stored at −80°C. Bones were cut on a cryostat until the BM was fully exposed and then harvested by melting the OCT. Open bones were blocked/permeabilized with 10% normal rabbit serum, 10% normal mouse serum, 10% Fc-block and 0.25% Triton X-100 for 2 hours at room temperature, stained with anti-GFP/YFP (Life Technologies), goat anti-NKp46 (AF2225, R&D Systems), anti-Ly6G (1A8, BD Biosciences), and anti-Ly6B2 (7/4, AbD Serotec) overnight at 4°C while shaking. Bones were then washed with PBS and incubated for 2 hours at room temperature in secondary antibody AF647 rabbit anti-goat IgG. After antibody staining, bones were washed and nuclei were stained with DAPI (Sigma-Aldrich). Stained bone was imaged in a chambered cover glass using a Leica TCS SP8 confocal microscope. Images were processed using Imaris Bitplane software.

Ex vivo Detection of IL-12p70 from Tissue Preparations

MesLN, spleen, and BM were minced and enzymatically digested at 37°C for 25 minutes in complete media (with the exception of FBS, which was excluded) supplemented with 50 μg/ml liberase TL (Roche) and 0.025% DNase I (Sigma-Aldrich) to improve extraction of DC populations. Following digestion, cell suspensions were generated from these tissues. Ten percent of the total cells from each tissue were cultured in 25 μl of complete medium for 18 hours and IL-12p70 was detected in the supernatant by Enhanced Sensitivity Cytometric Bead Array (BD Biosciences).

Supplementary Material

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2

ACKNOWLEDGEMENTS

This work was supported by the Division of Intramural Research of NIAID. We thank the NIAID animal facility staff, Dr. K Holmes and Dr. E Stregevsky, N Bubunenko, M Solano-Dias, J LeGrand, and K Beacht for technical assistance. We also thank Dr. AJ Radtke, Dr. M Guilliams, Dr. J Hettinger, and Dr. M Feuerer for technical advice and discussion. Dr. G Hart and Dr. EO Long graciously provided the protocol for NK depletion as well as purified antibody to perform this study. Dr. IE Brodsky generously provided Y. pseudotuberculosis. Additionally, we thank Dr. S Henri and Dr. S Tamoutounour for thoughtful discussions and critical reading of the manuscript.

Footnotes

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AUTHOR CONTRIBUTIONS

M.H.A., J.R.G., and Y.B. designed the studies. M.H.A., J.R.G., S.H., D.M.F., and C.W., performed the experiments and analyzed the data. A.L.B performed NanoString gene expression analysis. N.B., J.E.K., and T.W.H. provided valuable guidance and technical assistance. N.L.Q. and X.Z.S. helped design and perform the studies utilizing P. yoelii. G.T. assisted with designing and performing the NanoString gene expression analysis. M.H.A., J.R.G., and Y.B. wrote the manuscript.

Additional procedures can be found in the Supplemental Experimental Procedures.

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Supplementary Materials

1
NIHMS693740-supplement-1.pdf (23.4MB, pdf)
2
NIHMS693740-supplement-2.xlsx (132.2KB, xlsx)

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