Research ArticleINFECTIOUS DISEASES

Inflammatory monocytes hinder antiviral B cell responses

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Science Immunology  21 Oct 2016:
Vol. 1, Issue 4, eaah6789
DOI: 10.1126/sciimmunol.aah6789

B cells hoisted by their own petard: IFN-I

Certain pathogens, including HIV and hepatitis viruses, that lead to persistent infections are often associated with suboptimal antibody responses. Using lymphocytic choriomeningitis virus infection in mice, Fallet et al., Moseman et al., and Sammicheli et al. report that up-regulation of type I interferon (IFN-I) in the early phase of infection is a key contributor to premature deletion of virus-specific B cells. Blockade of IFN-I prevents B cell deletion. Although the studies agree that IFN-I does not act directly on B cells, they found that distinct immune cells mediate IFN-I–dependent deletion of B cells, depending on the system examined. Targeting of the IFN-I pathway could be used to restore B cell responses during persistent viral infections in humans.

Abstract

Antibodies are critical for protection against viral infections. However, several viruses, such as lymphocytic choriomeningitis virus (LCMV), avoid the induction of early protective antibody responses by poorly understood mechanisms. We analyzed the spatiotemporal dynamics of B cell activation to show that, upon subcutaneous infection, LCMV-specific B cells readily relocate to the interfollicular and T cell areas of draining lymph nodes, where they extensively interact with CD11b+Ly6Chi inflammatory monocytes. These myeloid cells were recruited to lymph nodes draining LCMV infection sites in a type I interferon– and CCR2-dependent fashion, and they suppressed antiviral B cell responses by virtue of their ability to produce nitric oxide. Depletion of inflammatory monocytes, inhibition of their lymph node recruitment, or impairment of their nitric oxide–producing ability enhanced LCMV-specific B cell survival and led to robust neutralizing antibody production. Our results identify inflammatory monocytes as critical gatekeepers that restrain antiviral B cell responses and suggest that certain viruses take advantage of these cells to prolong their persistence within the host.

INTRODUCTION

Antibodies (Abs) are critical for virus control and prevention of reinfection (1). Their production depends on B cells encountering viral antigens (Ags) in lymph nodes (LNs) draining infection sites, getting activated, interacting with different cells, proliferating, and differentiating into Ab-secreting cells. Each of these events occurs in distinct LN subcompartments, requiring the migration of B cells from niche to niche in a fast and tightly coordinated fashion (2). Thanks to the recent advent of multiphoton intravital microscopy (MP-IVM), several cellular and molecular events by which LNs orchestrate the generation of humoral immune responses have been clarified (35). However, how viral infections affect the spatiotemporal dynamics of B cell activation is not well defined. Moreover, the mechanisms whereby some viruses [e.g., lymphocytic choriomeningitis virus (LCMV)] interfere with the induction of early, potent neutralizing Ab responses remain largely unexplored.

Here, we used MP-IVM to study Ag-specific B cell behavior upon viral infection. We found that, upon LCMV infection, virus-specific B cells readily move from B cell follicles to the interfollicular and T cell areas of draining LNs, where they engage in prolonged interactions with and are eventually killed by a population of inflammatory monocytes that is recruited in a type I interferon (IFN-I)– and CCR2-dependent manner. Strategies aimed at preventing inflammatory monocyte accumulation within secondary lymphoid organs increased LCMV-specific B cell survival and caused robust neutralizing Ab production.

RESULTS

Spatiotemporal dynamics of B cell activation in response to vesicular stomatitis virus and LCMV infection

To begin addressing these issues, we infected mice subcutaneously into the hind footpad with either vesicular stomatitis virus (VSV) or LCMV—two viruses that have been widely used to study adaptive immune responses (1). Consistent with previous results obtained with systemic routes of infection (1), early, potent neutralizing Ab responses were induced upon local infection with VSV, but not with LCMV (Fig. 1A). Because the coevolution of the LCMV-mouse relationship might have resulted in the selection of a neutralizing epitope that is not readily recognized at sufficiently high avidity by germ line–encoded immunoglobulin (Ig) VH (variable region of Ig heavy chain)–VL (variable region of Ig light chain) region combinations in wild-type (WT) mice (1), we sought to correct for eventual disparities in the initial virus-specific B cell precursor frequency by making use of B cell receptor (BCR) transgenic mice. VSV-specific BCR transgenic mice (referred to as VI10YEN) have already been described (6); LCMV-specific BCR transgenic mice (referred to as KL25) were generated by crossing available LCMV-specific VH knock-in mice (6) with newly generated LCMV-specific VL transgenic mice (fig. S1A). The vast majority (~90%) of the resulting KL25 B cells bound to the LCMV glycoprotein (GP) and, upon in vitro incubation with LCMV, got readily activated and produced Abs to the same extent that VI10YEN B cells did in response to VSV (fig. S1, B to D). However, adoptive transfer of up to 107 KL25 B cells into DHLMP2A mice [which are devoid of surface-expressed and secreted Abs (7) but, in contrast to B cell–deficient mice, retain an intact LN architecture (8)] before subcutaneous LCMV infection did not result in a detectable neutralizing Ab response (Fig. 1B and fig. S2). By contrast, adoptive transfer of VI10YEN B cells using the same experimental setup—where Abs can be produced only by the transferred B cells—resulted in a readily detectable, potent neutralizing Ab response (Fig. 1B). Together, these results indicate that a low Ag-specific B cell precursor frequency is not the sole determinant of the impaired humoral immune response observed upon LCMV infection, and they suggest that events linked to LCMV replication in vivo actively interfere with the generation of a protective Ab response.

Fig. 1 Spatiotemporal dynamics of B cell activation in response to VSV and LCMV infection.

(A) Neutralizing Ab titers in the sera of C57BL/6 mice that were infected subcutaneously with 105 PFU of VSV (gray) or 105 FFU of LCMV (black) (n = 5). Results are representative of at least three independent experiments. (B) Neutralizing Ab titers in the sera of DHLMP2A mice that were transferred with 5 × 106 purified VI10YEN (gray) or KL25 (black) B cells 18 hours before subcutaneous infection with VSV or LCMV, respectively (n = 5). Results are representative of at least two independent experiments. (C) Multiphoton intravital (left and middle) or confocal (right) micrographs in the popliteal LN of a DHLMP2A mouse that was injected with VI10YEN (red) and WT (cyan) B cells 18 hours before subcutaneous VSVeGFP (green) infection. Snapshots were acquired at the indicated time points after infection. The broken white line denotes B cell follicles. Scale bars, 50 μm (left and middle) or 100 μm (right). Results are representative of at least five independent experiments. See also movie S2. (D) Multiphoton intravital (left and middle) or confocal (right) micrographs in the popliteal LN of a DHLMP2A mouse that was injected with KL25 (red) and WT (cyan) B cells 18 hours before subcutaneous LCMVeGFP (green) infection. Snapshots were acquired at the indicated time points after infection. The broken white line denotes B cell follicles. Scale bars, 50 μm (left and middle) or 100 μm (right). Results are representative of at least five independent experiments. See also movie S3. (E) Percentages of WT (blue) and VI10YEN (red) B cells within the indicated LN compartments 72 hours after VSV infection (n = 2). Results are representative of at least three independent experiments. See also movie S2. (F) Percentages of WT (blue) and KL25 (red) B cells within the indicated LN compartments 72 hours after LCMV infection (n = 2). Results are representative of at least three independent experiments. See also movie S3. (G) Meandering index of WT (blue) or KL25 (red) B cells in the IFA (total, left) or in IFAs that were >15 μm distant from GFP+ cells (GFP areas, right) 72 hours after LCMV infection (n = 2). Results are representative of at least two independent experiments. See also movie S3. Results are expressed as means ± SEM.

To gain insights into the mechanism whereby LCMV inhibits Ab responses, we next set out to directly visualize the spatiotemporal dynamics of B cell activation by performing MP-IVM in LNs draining infection sites. To this end, we infected mice subcutaneously into the right hind footpad with recombinant VSVeGFP (9, 10) or LCMV-M1eGFP (11, 12), both of which selectively drive green fluorescent protein (GFP) expression within infected cells and show replication kinetics in footpad-draining popliteal LNs that are very similar to the respective parental strains (fig. S3A). As previously reported (10), VSV replication in LNs was restricted to CD169+ subcapsular sinus (SCS) macrophages, and it reached peak levels at 8 to 12 hours after infection and ended within 24 to 48 hours (fig. S3, A and B). By contrast, LCMV replicated at early time points (12 hours after infection) in both SCS and medullary macrophages (12), peaked at 72 hours after infection where replication extended to stromal cells and myeloid cells in interfollicular areas (IFAs), and cleared in about a week (fig. S3, A and B). To visualize virus-specific B cells, we then adoptively transferred fluorescently labeled VI10YEN or KL25 B cells—along with WT polyclonal B cells—into DHLMP2A mice before subcutaneous infection [note that in uninfected mice, WT, VI10YEN, and KL25 B cells showed virtually identical motility parameters (i.e., speed, meandering index, and track direction); fig. S4, A and B, and movie S1]. We first analyzed the dynamics of B cell activation in response to VSV. Consistently with what has been previously reported with nonviral Ags or nonreplicating VSV (1315), as early as 2 hours after infection, Ag-specific B cells accumulated within the SCS, where they interacted extensively with infected CD169+ macrophages (Fig. 1C, fig. S4, C and D, and movie S2). By 8 hours after infection, despite residual viral replication within the SCS, VI10YEN B cells redistributed to the T cell/B cell border, where they remained until ~24 hours after infection (Fig. 1C, fig. S4, E and F, and movie S2). This redistribution was associated with robust VI10YEN B cell activation, as assessed by substantial up-regulation of CD25 and CD69 by 24 hours after infection (fig. S4G). By 72 hours after infection, VI10YEN B cells had undergone vigorous proliferation and had relocated back to the follicle (Fig. 1, C and E, and movie S2). We next analyzed the spatiotemporal dynamics of B cell activation in response to LCMV. LCMV replication in popliteal LNs was undetectable at 2 hours after infection, and, accordingly, no redistribution of Ag-specific B cells to the SCS occurred (Fig. 1D, fig. S5, A and B, and movie S3). At 8 hours after infection, we started to detect some LCMV-infected cells, but no substantial changes in the behavior of KL25 B cells occurred (fig. S5, C and D, and movie S3). By contrast, at 24 hours after infection, there was an extensive redistribution of KL25 B cells to the IFA, with a fraction of Ag-specific B cells engaging in interactions with virus-replicating (GFP+) cells (Fig. 1D, fig. S5E, and movie S3). Whereas the meandering index of the few remaining follicular KL25 B cells was indistinguishable from that of control WT B cells, KL25 B cells that relocated to the IFA showed confined motility, characterized by a low meandering index and restricted directionality (fig. S5, F to H, and movie S3). By contrast, the few control WT B cells that ended up in the IFA readily moved back to the follicle (fig. S5H). The abovementioned changes in motility parameters were associated with robust KL25 B cell activation, as assessed by significant up-regulation of CD25 and CD69 by 24 hours after infection (fig. S5I). Together, these results indicate that LCMV-specific B cells recognize LCMV and are activated in vivo, yet they fail to produce Abs (Fig. 1B). At 72 hours after infection, when VI10YEN B cells in VSV-infected mice have already returned to B cell follicles (Fig. 1, C and E, and movie S2), the vast majority of KL25 B cells in LCMV-infected mice were still confined to the IFA (Fig. 1, D and F, and movie S3). The different localization of virus-specific B cells in VSV- and LCMV-infected mice correlated with a differential expression of the B cell follicle-homing chemokine receptor CXCR5 (fig. S5J). Because KL25 B cells at the IFA showed confined motility, particularly in areas where LCMV-replicating cells (GFP+) were absent (Fig. 1G), we reasoned that LCMV-specific B cells actively engaged with a population of cells that impaired their differentiation into Ab-secreting cells.

Inflammatory monocytes, recruited to LNs in an IFN-I– and CCR2-dependent fashion, suppress LCMV-specific Ab responses

Among potential immunosuppressive cells, we focused on CD11b+ myeloid cells because they are recruited to LNs draining infection sites (16), and subsets of these cells have been shown to suppress adaptive (i.e., T cell–mediated) immune responses in various pathophysiological settings (1619). We therefore set out to characterize the CD11b+ cell population that is recruited to popliteal LNs upon footpad LCMV infection. We found that a distinct population of CD11b+Ly6ChiF4/80+MHC-IIintCD11clo/int inflammatory (or “classical”) monocytes (20) was recruited to draining LNs as early as 24 hours after LCMV infection and persisted for at least 7 days after infection (Fig. 2, A and B, and fig. S6A). To determine whether these inflammatory monocytes played a role in regulating LCMV-specific B cell responses, we designed experimental conditions to inhibit their LN recruitment or to deplete them. Because IFN-I and CCR2 have been shown to regulate inflammatory monocyte recruitment to infected or inflamed tissues (2123), we reasoned that these molecules might also be implicated in the LN recruitment of CD11b+Ly6Chi cells upon LCMV infection. Inflammatory monocytes were not recruited in the LNs of LCMV-infected type I IFN receptor (IFNAR)−/− and CCR2−/− mice, and they were absent in the LNs of inflammatory monocyte–depleted animals treated with anti–Gr-1 Abs (Fig. 2C and fig. S6B). KL25 B cells that were transferred into IFNAR−/−, CCR2−/−, or anti–Gr-1–treated mice produced large quantities of LCMV-GP–binding and LCMV-neutralizing Abs (Fig. 2, D and E). LCMV-specific Ab responses in the absence of inflammatory monocytes were also detected (albeit at substantially lower levels) in a more physiological setting that did not entail the adoptive transfer of KL25 B cells (Fig. 2F). These results, based on three mechanistically distinct approaches, indicate that inflammatory monocytes prevent the induction of LCMV-specific Ab responses. One potential caveat of the experimental strategies that we used to prevent inflammatory monocyte accumulation within LNs is that they might have increased LCMV titers and, therefore, the amount of Ags available to LCMV-specific B cells. Consistently with previously published literature (24), IFNAR−/− mice were characterized by higher viral titers in draining LNs at the peak (72 hours after infection; see fig. S3A) of LCMV replication (fig. S6C). However, we did not detect increased viral titers at 72 hours after infection in LNs of CCR2−/− or anti–Gr-1–treated mice (fig. S6C). Moreover, infection of WT mice with up to 100-fold higher doses of LCMV did not lead to LCMV-specific Ab responses (fig. S7). Together, these results suggest that the mechanism whereby inflammatory monocytes hinder Ab responses upon LCMV infection of WT mice does not rely on limiting Ag availability to B cells. To further increase the specificity of our approaches targeting inflammatory monocytes and to rule out potential off-target effects, we next reconstituted CCR2−/− mice with WT monocytes. Adoptive transfer of 5 × 106 bone marrow (BM)–derived monocytes from WT mice into CCR2−/− animals 6 hours after LCMV infection resulted in the LN recruitment of inflammatory monocytes and in the inhibition of LCMV-specific Ab responses (Fig. 2G). The induction of neutralizing Abs that we observed in the absence of inflammatory monocytes had a significant impact on viral clearance because CCR2−/− mice had lower LCMV titers than WT mice at day 7 after infection (Fig. 2H), despite T cell responses that were, if anything, lower in CCR2−/− than in WT mice (25).

Fig. 2 Inflammatory monocytes, recruited to LNs in an IFN-I– and CCR2-dependent fashion, suppress LCMV-specific Ab responses.

(A) FACS plots showing CD11b and Ly6C expression in the popliteal LNs of mice injected in the footpad with PBS (left) or LCMV (right) 72 hours earlier. Numbers show the percentage of cells within the gates. Plots are representative of at least 10 independent experiments. (B) Number of CD11b+Ly6Chi cells within the popliteal LNs of WT mice at the indicated time points after subcutaneous LCMV infection. Results are representative of at least three independent experiments. (C) Number of CD11b+Ly6Chi cells within the popliteal LNs of WT (black), IFNAR−/− (red), CCR2−/− (blue), or anti–Gr-1–treated WT mice (green) 72 hours after subcutaneous LCMV infection. (D and E) KL25 B cells (5 × 106) were transferred into WT (black), IFNAR−/− (red), or CCR2−/− (blue) mice or into WT mice injected with anti–Gr-1 (green) or isotype control (black) Abs before subcutaneous LCMV infection. LCMV-GP–binding (D) or neutralizing (E) Abs were measured in the sera 7 days after LCMV infection. LCMV-GP–binding Abs are expressed as fold induction (F.I.) over uninfected controls. Results are representative of at least five independent experiments. (F) LCMV-GP–binding Abs in the sera of WT (black), IFNAR−/− (red), or CCR2−/− (blue) mice or anti–Gr-1–treated WT mice (green) 7 days after subcutaneous LCMV infection. Data are expressed as F.I. over uninfected controls. Results are representative of two independent experiments. (G) KL25 B cells were transferred into WT (black), CCR2−/− (blue), or CCR2−/− mice reconstituted with 5 × 106 WT monocytes (yellow). LCMV-GP–binding Abs (F.I. over uninfected controls) were measured 7 days after subcutaneous LCMV infection. Results are representative of at least two independent experiments. (H) LCMV titers 7 days after infection in popliteal LNs of WT (black) or CCR2−/− (blue) mice that were transferred with 5 × 106 KL25 B cells before subcutaneous LCMV infection. Results are representative of at least two independent experiments. Results are expressed as means ± SEM.

Inflammatory monocytes recruited to LCMV-infected LNs interact with Ag-specific B cells and impair their survival

We next sought to characterize how inflammatory monocytes recruited to LNs draining LCMV infection sites hindered Ab responses. Because inflammatory monocytes are characterized by the concomitant expression of the chemokine receptors CX3CR1 and CCR2 (20, 26), we crossed CX3CR1GFP/+ (27) with CCR2RFP/+ (28) reporter mice to visualize this cell population in LCMV-draining LNs. Whereas uninfected CX3CR1GFP/+;CCR2RFP/+ mice had very few LN cells that expressed both GFP and RFP (red fluorescent protein), LCMV infection induced a prominent accumulation of GFP+RFP+ in LN IFAs (Fig. 3A and fig. S8, A to C). Note that most (~51%) of GFP+RFP+ cells within LNs of infected CX3CR1GFP/+;CCR2RFP/+ mice were CD11b+Ly6Chi monocytes, and, conversely, virtually all CD11b+Ly6Chi cells expressed both GFP and RFP (fig. S8, A and B).

Fig. 3 Inflammatory monocytes recruited to LCMV-infected LNs interact with Ag-specific B cells and impair their survival.

(A) Confocal micrographs in the popliteal LNs of CX3CR1GFP/+;CCR2RFP/+ mice that were transferred with fluorescent WT (cyan) and KL25 (red) B cells, injected subcutaneously with either PBS (left) or LCMV (right) and killed 72 hours after infection. Scale bars, 100 μm. The broken white line denotes B cell follicles. Results are representative of at least three independent experiments. (B) Multiphoton intravital micrograph in the popliteal LNs of an LCMV-infected CX3CR1GFP/+;CCR2RFP/+ mouse that was transferred with fluorescent WT (cyan) and KL25 (red) B cells and killed 72 hours after infection. Scale bar, 20 μm. The result is representative of at least three independent experiments. See also movie S4. (C) Quantification of the contact time (in minutes) between CXCR1+CCR2+ cells and WT (blue) or KL25 (red) B cells in the popliteal LNs of CX3CR1GFP/+;CCR2RFP/+ mice that were infected subcutaneously with LCMV 72 hours earlier. See also movie S4 (n = 4). Results are representative of at least two independent experiments. (D and E) KL25 B cells (5 × 106) were transferred into WT (black), IFNAR−/− (red), CCR2−/− (blue), or WT mice injected with anti–Gr-1 (green) or isotype control (black) Abs and infected subcutaneously with LCMV. Frequency of annexin V+ apoptotic KL25 B cells (D) and number of KL25 B cells (E) were quantified in the popliteal LNs 3 days after infection. Results are representative of at least five independent experiments. (F) VI10YEN B cells (5 × 106) were transferred into WT mice before subcutaneous immunization with PFA-inactivated VSV and either simultaneously infected (red) or not (black) with LCMV. VI10YEN B cells were quantified in the popliteal LN 72 hours after immunization. Results are representative of at least two independent experiments. Results are expressed as means ± SEM.

Because inflammatory monocytes within LNs of LCMV-infected mice were found to almost completely colocalize with LCMV-specific B cells (Fig. 3A), we hypothesized that inflammatory monocytes might physically interact with Ag-specific B cells to suppress Ab production. To test this hypothesis, we performed MP-IVM in popliteal LNs of CX3CR1GFP/+;CCR2RFP/+ mice that were adoptively transferred with fluorescent WT and KL25 B cells before LCMV infection. As shown in movie S4 and Fig. 3 (B and C), inflammatory monocytes engaged in prolonged interaction with LCMV-specific B cells but not with control WT B cells. The notion that such interactions are responsible for the confined motility of LCMV-specific B cells within LN IFAs of WT mice is further demonstrated by the observation that LCMV-specific B cells had a higher meandering index when transferred to CCR2−/− mice than to WT mice (fig. S9). We then asked whether the physical interaction of inflammatory monocytes with LCMV-specific B cells affected B cell survival. A lower frequency of apoptotic cells was detected within KL25 B cells isolated from the LNs of IFNAR−/−, CCR2−/−, or anti–Gr-1–treated mice as compared with KL25 B cells isolated from the LNs of WT mice (Fig. 3D); accordingly, depletion of inflammatory monocytes or inhibition of their LN recruitment led to a higher recovery of KL25 B cells in the popliteal LNs of LCMV-infected mice (Fig. 3E). In contrast to the notable effect on KL25 B cell survival, inflammatory monocytes did not significantly affect KL25 B cell proliferation or early activation phenotype upon LCMV infection (fig. S10). We next asked whether the detrimental effect that inflammatory monocytes recruited to LCMV-infected LNs exerted on B cells was dependent on the Ag specificity of B cells. To this end, we sought to analyze VSV-specific B cell responses in LCMV-infected mice. Because the IFN-I produced in response to LCMV might affect VSV titers (and hence the amount of viral Ags available for B cells) in coinfection experiments, we decided to challenge LCMV-infected mice with paraformaldehyde (PFA)–inactivated VSV. As shown in Fig. 3F, fewer VI10YEN B cells were recovered in LCMV-infected mice concomitantly challenged with PFA-inactivated VSV 3 days earlier when compared with mice immunized with inactivated VSV alone. Together, these results indicate that inflammatory monocytes recruited to LNs draining an LCMV infection site interact with activated B cells regardless of their Ag specificity and induce B cell apoptosis.

We next asked whether VSV induced LN recruitment of inflammatory monocytes and, if so, whether these cells suppressed Ag-specific B cell responses in that setting as well. CD11b+Ly6Chi monocytes were recruited to VSV-infected LNs within 24 hours after infection, but they disappeared by 48 to 72 hours after infection (fig. S11A), likely because CCL-2 expression within VSV-infected LNs was not sustained (fig. S11B). Possibly as a result of this rapid clearance, CD11b+Ly6Chi cells recruited to VSV-infected LNs did not suppress VSV-specific B cell responses because VSV-infected DHLMP2A mice injected with VI10YEN showed no differences in VSV-specific B cell recovery or in neutralizing Ab titers when treated with anti–Gr-1 or isotype-matched Abs (fig. S11, C to E).

Inflammatory monocytes inhibit B cell responses via nitric oxide

Next, we sought to characterize the mechanism whereby inflammatory monocytes recruited to LNs draining LCMV infection sites hinder B cell responses. We first checked whether T cells were required for monocyte-induced B cell suppression. In contrast to the well-established role that both CD4+ and CD8+ T cells play in influencing LCMV-specific B cell responses at late time points after infection (2933), depletion of either CD4+ or CD8+ cells did not alter KL25 B cell recovery 3 days after infection (fig. S12, A and B), suggesting that inflammatory monocyte–mediated B cell suppression occurs independently of CD4+ and CD8+ T cells. We then set out to identify the molecular mediator of monocyte-mediated B cell inhibition. We focused on nitric oxide, a well-established myeloid cell–derived immunosuppressive factor (18, 34). To create a system in which CCR2+ inflammatory monocytes that accumulate within LCMV-infected LNs (but not CCR2 cells) lacked the capacity to produce nitric oxide, we reconstituted irradiated WT recipients with a 1:1 mixture of iNOS (inducible nitric oxide synthase)−/− and CCR2−/− BM. Irradiated WT recipients that were reconstituted with 100% WT BM, 100% CCR2−/− BM, or a 1:1 mixture of WT and CCR2−/− BM served as controls. Nitric oxide did not affect the capacity of inflammatory monocytes to accumulate within LCMV-infected LNs, but it severely impaired their B cell–suppressive ability (Fig. 4, A to C). Together, these results indicate that inflammatory monocytes hinder B cell responses via nitric oxide.

Fig. 4 Inflammatory monocytes inhibit B cell responses via nitric oxide.

Irradiated WT recipients were reconstituted with WT (black), CCR2−/− (blue), a 1:1 mixture of WT and CCR2−/− (gray), and a 1:1 mixture of iNOS−/− and CCR2−/− (orange) BM 8 weeks before injection with 5 × 106 KL25 B cells, followed by subcutaneous LCMV infection. (A) Number of CD11b+Ly6Chi cells recovered in the popliteal LNs 3 days after infection. (B) Number of KL25 B cells recovered in the popliteal LNs 3 days after infection. (C) Serum LCMV-GP–binding Abs (F.I. over uninfected controls) measured 7 days after infection. Data are representative of at least two independent experiments. Results are expressed as means ± SEM.

DISCUSSION

In this study, we have analyzed the spatial and temporal constraints whereby virus-specific naïve B cells respond to infections with live lymph-borne viruses that either do (VSV) or do not (LCMV) elicit early, robust neutralizing Ab responses. Whereas the spatiotemporal dynamics of early B cell activation in response to live VSV follows what has been previously described with nonreplicating Ags (1315, 35), upon LCMV infection, Ag-specific B cells readily relocate to the IFA and T cell area of draining LNs, where they extensively interact with CD11b+Ly6Chi inflammatory monocytes that are recruited in an IFN-I– and CCR2-dependent manner. The main consequence of such interaction is impaired B cell survival, caused by nitric oxide produced by inflammatory monocytes. Depletion of inflammatory monocytes, inhibition of their LN recruitment, or deficiency in their nitric oxide production improves LCMV-specific B cell survival and leads to robust neutralizing Ab production.

Our results shed light on a long-standing question in viral immunobiology, which is how noncytopathic viruses such as LCMV avoid the induction of early, effective neutralizing Ab responses (1). The mechanism constraining the early B cell response to LCMV described here has significant functional consequences in terms of viral clearance because mice devoid of inflammatory monocytes have >50-fold lower LCMV titers than WT mice 7 days after infection. IFN-I receptor blockade had a larger effect on B cell responses than CCR2 blockade or Gr-1+ cell depletion, suggesting that LN recruitment of inflammatory monocytes is only one of the mechanisms whereby IFN-I impairs antiviral Ab responses. The identification of additional IFN-I–dependent mechanisms limiting humoral immune responses requires further investigation.

Although the capacity of myeloid-derived cells to suppress T cell responses is being increasingly recognized (1719), very little is known on myeloid cell–mediated B cell inhibition. The results reported here unravel a key role for inflammatory monocytes in limiting humoral responses and pave the road for further investigations aimed at exploring whether similar suppressive mechanisms are operative in other infections or in the tumor microenvironment. The mechanism whereby inflammatory monocytes engage in prolonged interactions with activated B cells is still unresolved. One possibility is that these cells capture and display surface-bound viral particles [in a way reminiscent of what has been previously described for SCS macrophages (15)].

Experimental needs pertaining to intravital microscopy studies required the adoptive transfer of supraphysiological numbers of high-affinity virus-specific BCR transgenic cells, which raises the question of whether the immunosuppressive mechanism described here is operative for B cells expressing BCRs with lower affinity. Although the notion that inflammatory monocytes suppress LCMV-specific Ab responses in WT mice as well—where, presumably, BCRs with a wide range of affinities for LCMV are present—suggests that this is a general phenomenon that occurs independently of BCR signal strength, further investigation is needed to definitively settle this issue.

Whether the suppressive effect of monocyte-derived nitric oxide on B cells is direct or indirect remains to be clarified. Nitric oxide produced by myeloid-derived suppressor cells has been shown, under certain experimental conditions, to inhibit T cell responses (17, 18). We ruled out a role for CD4+ or CD8+ T cells in the B cell response to LCMV at day 3 after infection (the time point where the effect of inflammatory monocytes on B cell responses is already evident), suggesting that inflammatory monocyte–mediated B cell suppression is T cell–independent. Although these data, together with the reported extensive interactions between monocytes and activated B cells, argue in favor of monocytes directly inhibiting B cells, further investigation is warranted.

The results presented here establish inflammatory monocytes as critical gatekeepers that prevent antiviral B cell responses. From an evolutionary perspective, it is tempting to speculate that viruses such as LCMV might have evolved strategies to exploit the recruitment of these cells to LNs draining infection sites to prolong their persistence within the host and to maximize the chance of being transmitted to other susceptible individuals.

MATERIALS AND METHODS

Mice

C57BL/6 and CD45.1 (inbred C57BL/6) mice were purchased from Charles River Laboratories. DHLMP2A mice (7) (inbred Balb/c) were originally provided by K. Rajewsky (Harvard Medical School) and bred more than 10 generations against C57BL/6 mice. Heavy-chain knock-in and light-chain BCR transgenic mice specific for VSV Indiana [VI10YEN (6)] and heavy-chain knock-in BCR transgenic mice specific for LCMV WE [KL25 (6)] were obtained through the European Virus Archive. Light-chain BCR transgenic mice specific for LCMV WE (inbred C57BL/6) were generated as described in the legend to fig. S1A and bred against heavy-chain BCR transgenic mice specific for LCMV WE before subsequent use (except in selected experiments where Ab titers against LCMV were measured). β-Actin–DsRed [B6.Cg-Tg(CAG-DsRed*MST)1Nagy/J] and β-actin–CFP (cyan fluorescent protein) [B6.129(ICR)-Tg(CAG-ECFP)CK6Nagy/J] transgenic mice were purchased from the Jackson Laboratory. For imaging experiments, VI10YEN and KL25 mice were bred against both β-actin–DsRed and β-actin–CFP mice. Mice lacking the IFN-I receptor (IFNAR1−/− mice) were obtained through the Swiss Immunological Mutant Mouse Repository (Zurich, Switzerland). CCR2−/− (B6.129S4-Ccr2tm1Ifc/J) mice were purchased from the Jackson Laboratory. CX3CR1GFP/+ (27) and CCR2RFP/+ (28) mice were provided by I. Charo (University of California, San Francisco) by way of B. Engelhardt (University of Bern). BM from iNOS−/− mice was provided by E. Clementi (University of Milan). BM chimeras were generated by irradiation of C57BL/6 mice with ~730 rad and reconstitution with the indicated BM; mice were allowed to reconstitute for at least 8 weeks before use. The mice were housed under specific pathogen–free conditions and used at 8 to 10 weeks of age, unless otherwise indicated. In all experiments, mice were matched for age and sex before experimental manipulation. All experimental animal procedures were approved by the Institutional Animal Committee of the San Raffaele Scientific Institute.

Infections and immunizations

Mice were infected intrafootpad with the following: 1 × 105 plaque-forming units (PFU) of VSV serotype Indiana; 1 × 105 PFU of VSVeGFP (9, 10); 1 × 105, 1 × 106, or 1 × 107 focus-forming units (FFU) of LCMV-M1 (a variant of LCMV clone 13 that is recognized by KL25 B cells) (11); or 1 × 105 FFU of LCMV-M1eGFP. Alternatively, mice were immunized intrafootpad with 10 μg of recombinant LCMV WE GP-1–human IgG fusion protein (36) mixed (at a ratio of 1:1) either with a squalene-based oil-in-water nanoemulsion (AddaVax, InvivoGen) or with aluminum potassium sulfate dodecahydrate (Sigma). Viruses were propagated and quantified, as described previously (37, 38), and dissolved in 20 μl of phosphate-buffered saline (PBS) before footpad injection.

Mice were retro-orbitally bled at the indicated time points for VSV- or LCMV-specific Abs measured by VSV neutralization assay (37), LCMV focus reduction assay (39), or LCMV GP-1–binding enzyme-linked immunosorbent assay (ELISA) (40). For determination of VSV- or LCMV-neutralizing IgG titers, sera were incubated with equal volumes of 0.1 M 2-mercaptoethanol in Dulbecco’s modified Eagle’s medium with 2% fetal calf serum for 1 hour at room temperature before dilution (41). Viral titers from sera, LNs, and spleens of VSV- or LCMV-infected mice were measured by plaque (8) or focus assay (39), respectively. All infectious work was performed in designated Biosafety Level 2 (BSL-2) and BSL-3 workspaces in accordance with institutional guidelines.

B cell activation in vitro

Naïve B cells from spleens of VI10YEN and KL25 mice were negatively selected by magnetic isolation with CD43 beads (Miltenyi Biotec), as described previously (37). The purity was ~98%, as determined by B220 staining. Purified B cells (106 cells/ml) were cultured in the presence of PFA-inactivated VSV or LCMV (0.5 multiplicity of infection equivalent). In some experiments, B cells were harvested after 24 hours and analyzed by flow cytometry for the surface expression of activation markers (see below). In other experiments, B cells were cultured for 72 hours, and the supernatants were tested for the presence of total mouse IgM by ELISA.

Adoptive transfer experiments

Naïve B cells from spleens of C57BL/6, VI10YEN, VI10YEN × β-actin–DsRed, VI10YEN × β-actin–CFP, KL25, KL25 × β-actin–DsRed, and KL25 × β-actin–CFP mice were negatively selected by magnetic isolation with CD43 beads (Miltenyi Biotec), as described previously (37). The purity was ~98%, as determined by B220 staining. B cells (1 × 106, 5 × 106, or 1 × 107) were injected intravenously into the indicated recipient animals 18 hours before footpad virus infection. In selected imaging experiments, WT, VI10YEN, and KL25 B cells were labeled with 10 μM CMAC (7-amino-4-chloromethylcoumarin), 10 μM CMTMR [(5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethylrhodamine)], 2.5 μM BODIPY 650/665, or 1 μM Deep Red (Life Technologies) for 20 min at 37°C in plain RPMI before adoptive transfer, as described previously (42). In some experiments, inflammatory monocytes were depleted by intravenous injection of 200 μg of anti–Gr-1 (RB6-8C5, Bio X Cell) Abs every 48 hours, beginning 1 day before footpad virus infection. In some experiments, CD4+ T cells were depleted by intravenous injection of 200 μg of anti-CD4 (GK1.5, Bio X Cell) Abs 3 days and 1 day before infection, whereas in other experiments, CD8+ T cells were depleted by intravenous injection of 200 μg of anti-CD8 (YTS 169.4, Bio X Cell) Abs 1 day before and 3 days after infection. For interleukin-10 receptor (IL-10R)–blocking experiments, 500 μg of anti–IL-10R (1B1-3a, Bio X Cell) Abs was injected intravenously 6 hours after footpad infection. For monocyte reconstitution experiments, monocytes from the BM of C57BL/6 or CX3CR1-GFP;CCR2-RFP mice were negatively selected by magnetic isolation using the Monocyte Isolation Kit (BM) (Miltenyi Biotec), according to the manufacturer’s instructions. The purity was >95%, as determined by CD11b and Ly6C staining. Monocytes (5 × 106) were injected intravenously into CCR2−/− mice 6 hours after footpad infection.

Cell isolation and flow cytometry

Single-cell suspensions of spleens and LNs were generated, as described previously (37). All flow cytometry stainings of surface-expressed and intracellular molecules were performed, as described previously (37, 42). Fluorochrome-conjugated Abs to B220 (RA3-6B2), CD45.1 (A20), CD45.2 (104), CD69 (H1.2F3), CD25 (PC61), CD11b (M1/70), Ly6G (IA8), Ly6C (HK1.4), F4/80 (BM8), CD11c (N418), MHC-II (major histocompatibility complex II) (I-Ab, AF6-120.1), CD4 (RM4-5), CD8 (53-6.7), CXCR5 (2G8), IgD (11-26c.2a), CD80 (16-10A1), IFN-γ (XMG1.2), and TNF-α (tumor necrosis factor–α) (MP6-XT22) were purchased from BioLegend, eBioscience, or BD Pharmingen. B cell proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester dilution, as described previously (10). Intracellular IFN-γ staining was performed in the presence or absence of GP61 or GP33, as described previously (38). The proportion of LCMV-binding B cells in WT or KL25 mice was assessed by virtue of their ability to bind to the recombinant LCMV WE GP-1–human IgG fusion protein (36), using the Strep-tag/Strep-Tactin system (IBA), according to the manufacturer’s instructions. All flow cytometry analyses were performed in fluorescence-activated cell sorting (FACS) buffer containing PBS with 2 mM EDTA and 2% fetal bovine serum on either a FACSCanto or a FACS Fortessa (BD Pharmingen) and analyzed with FlowJo software (TreeStar).

Confocal immunofluorescence histology

Confocal microscopy analysis of popliteal LNs was performed, as described previously (37). The following primary Abs were used for staining: rat anti-B220 (RA3-6B2), rat anti-CD169 (AbD Serotec), rabbit anti-GFP (Invitrogen), rat anti-LCMV nucleoprotein (VL-4, Bio X Cell), and rat anti-TCRβ (T cell receptor β) (H57-597, BioLegend). The following secondary Abs were used for staining: Alexa Fluor 488–, Alexa Fluor 514–, Alexa Fluor 568–, or Alexa Fluor 647–conjugated goat anti-rabbit or anti-rat IgG (Life Technologies), and Alexa Fluor 647–conjugated chicken anti-rat IgG (Life Technologies). Images were acquired on an inverted Leica microscope (SP8, Leica Microsystems) with a motorized stage for tiled imaging. To minimize fluorophore spectral spillover, we used the Leica sequential laser excitation and detection modality. B cell follicles, IFAs, and T cell areas were defined on the basis of the positioning of naïve B cells and, in certain experiments, by B220 and TCRβ stainings, as described previously (12).

Statistical analyses

Results are expressed as means ± SEM. All statistical analyses were performed in Prism 5 (GraphPad Software). Means between two groups were compared with unpaired two-tailed t test. Means among three or more groups were compared with one-way or two-way analysis of variance with Bonferroni posttest.

SUPPLEMENTARY MATERIALS

immunology.sciencemag.org/cgi/content/full/1/4/eaah6789/DC1

Methods

Fig. S1. Generation and characterization of LCMV-specific light-chain transgenic mice.

Fig. S2. KL25 B cells do not produce LCMV-neutralizing Abs in vivo upon LCMV infection, regardless of the number of cells transferred.

Fig. S3. VSV and LCMV replication in popliteal LNs upon footpad infection.

Fig. S4. Spatiotemporal dynamics of B cell activation upon VSV infection.

Fig. S5. Spatiotemporal dynamics of B cell activation upon LCMV infection.

Fig. S6. Phenotype, recruitment determinants, and effect on viral titers of CD11b+Ly6Chi monocytes.

Fig. S7. KL25 B cells do not produce LCMV-neutralizing Abs in vivo upon LCMV infection, regardless of the virus dose.

Fig. S8. Characterization of CD11b+Ly6Chi monocytes in CX3CR1GFP/+;CCR2RFP/+ reporter mice.

Fig. S9. Inflammatory monocytes restrict LCMV-specific B cell motility in the IFA.

Fig. S10. KL25 B cell phenotype upon adoptive transfer into LCMV-infected WT or CCR2−/− mice.

Fig. S11. Inflammatory monocytes are recruited only transiently to VSV-infected LNs and do not suppress B cell responses.

Fig. S12. T cells are not required for inflammatory monocyte–mediated B cell suppression.

Table S1. Excel file containing tabulated data for Figs. 1 to 4 and figs. S1 to S12.

Movie S1. WT, VI10YEN, and KL25 B cells show indistinguishable motility in uninfected mice.

Movie S2. Spatiotemporal dynamics of B cell activation upon footpad VSV infection.

Movie S3. Spatiotemporal dynamics of B cell activation upon footpad LCMV infection.

Movie S4. Inflammatory monocytes recruited to LCMV-infected LNs interact with Ag-specific B cells.

References (43, 44)

REFERENCES AND NOTES

Acknowledgments: We thank R. Serra and M. Silva for secretarial assistance; A. Fiocchi and M. Raso for technical assistance; I. Charo and B. Engelhardt for CX3CR1GFP/+;CCR2RFP/+ mice; E. Clementi for iNOS−/− BM; M. Perro and U. H. von Andrian for help with the setup of the popliteal LN surgical preparation for intravital microscopy; F. V. Chisari, P. Dellabona, and R. Pardi for critical reading of the manuscript; and D. D. Pinschewer and all the members of the Iannacone laboratory for helpful discussions. Confocal immunofluorescence histology was carried out at Alembic, an advanced microscopy laboratory established by the San Raffaele Scientific Institute and the Vita-Salute San Raffaele University. Flow cytometry was carried out at FRACTAL, a flow cytometry resource and advanced cytometry technical applications laboratory established by the San Raffaele Scientific Institute. Funding: This work was supported by European Research Council grants 281648 (to M.I.) and 250219 (L.G.G.), Italian Association for Cancer Research (AIRC) grants 9965 and 15350 (to M.I.), Italian Ministry of Health grant GR-2011-02347925 (to M.I.), European Molecular Biology Organization Young Investigator Program (to M.I.), a Marie Curie Co-funding of Regional, National, and International Programmes (COFUND) (INVEST) fellowship for experienced researchers (to M.K.), and a Career Development Award from the Giovanni Armenise–Harvard Foundation (to M.I.). Author contributions: S.S. and M.K. designed and performed the experiments, analyzed the data, prepared the figures, and wrote the paper. P.D.L., N.J.d.O., M.D.G., J.F., C.C., and C.G.M. performed the experiments and analyzed the data. B.F. and J.C.d.l.T. provided the reagents. L.G., L.S., and M.M. provided technical assistance. R.O. performed the gene expression profile analyses. K.L. and P.D.G. generated the KL25 light-chain transgenic mice. L.G.G. provided conceptual advice and revised the paper. M.I. designed and coordinated the study, provided funding, analyzed the data, and wrote the paper. S.S. and M.K. performed the statistical analyses. Competing interests: The authors declare that they have no competing interests. Data and materials availability: KL25 light-chain transgenic mice used in the study were generated in the laboratory of P.D.G. Requests for this strain should be addressed to P.D.G. (pgreen{at}u.washington.edu) and M.I. (iannacone.matteo{at}hsr.it).
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