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Inflammatory monocytes are detrimental to the host immune response during acute infection with Cryptococcus neoformans

Cryptococcus neoformans is a leading cause of invasive fungal infections among immunocompromised patients. However, the cellular constituents of the innate immune response that promote clearance versus progression of infection upon respiratory acquisition of C. neoformans remain poorly defined. In this study, we found that during acute C. neoformans infection, CCR2+ Ly6Chi inflammatory monocytes (IM) rapidly infiltrate the lungs and mediate fungal trafficking to lung-draining lymph nodes. Interestingly, this influx of IM is detrimental to the host, since ablating IM or impairing their recruitment to the lungs improves murine survival and reduces fungal proliferation and dissemination. Using a novel conditional gene deletion strategy, we determined that MHC class II expression by IM did not mediate their deleterious impact on the host. Furthermore, although ablation of IM reduced the number of lymphocytes, innate lymphoid cells, and eosinophils in the lungs, the effects of IM were not dependent on these cells. We ascertained that IM in the lungs upregulated transcripts associated with alternatively activated (M2) macrophages in response to C. neoformans, consistent with the model that IM assume a cellular phenotype that is permissive for fungal growth. We also determined that conditional knockout of the prototypical M2 marker arginase 1 in IM and deletion of the M2-associated transcription factor STAT6 were not sufficient to reverse the harmful effects of IM. Overall, our findings indicate that C. neoformans can subvert the fungicidal potential of IM to enable the progression of infection through a mechanism that is not dependent on lymphocyte priming, eosinophil recruitment, or downstream M2 macrophage polarization pathways. These results give us new insight into the plasticity of IM function during fungal infections and the level of control that C. neoformans can exert on host immune responses.


Introduction
The ubiquitous encapsulated yeast Cryptococcus neoformans causes invasive fungal infections in immunocompromised patients, particularly those with AIDS, solid organ transplants, and cancer [1]. Even with optimal combination antifungal therapy, cryptococcosis has a high morbidity and mortality rate [2,3]. Since C. neoformans enters the respiratory tract before disseminating to the central nervous system [1], defining the cellular and molecular mechanisms of the pulmonary innate immune response is critical for the development of novel treatment options that can promote fungal sterilization in the lungs.
The role of IM in innate immunity to acute infection with C. neoformans has not been systematically examined. In models of subacute and chronic pulmonary cryptococcosis, there is an initial fungal expansion phase that is followed by prolonged, but largely progressive, fungal clearance through CD4 + and CD8 + T cell-dependent mechanisms [9,10]. During the expansion phase, CCR2-dependent signals mediate the accumulation of DCs and macrophages in the lungs [11][12][13]. The latter express inducible nitric oxide synthase (NOS2) and tumor necrosis factor (TNF) and appear to act in a fungicidal manner in vitro [13]. Defective CCR2 signaling impairs fungal clearance and correlates with the development of T helper 2 (Th2) cytokine-dominated responses [14,15]. More recent work indicates that IM and macrophages may play important roles in protective immune responses generated by candidate vaccine strains of C. neoformans [16][17][18][19]. These data suggest that IM and their derivatives may play beneficial roles in innate immunity during subacute and chronic cryptococcal infections.
On the other hand, in a lethal model of acute cryptococcosis, it has been observed that enhanced IM accumulation in the lungs correlates with decreased survival [20]. It is not known if IM may promote progressive infection by specific effects on fungal growth or T helper responses or if IM influx is part of a general inflammatory response in the lungs during acute cryptococcal infection. C. neoformans is also a facultative intracellular pathogen that has been shown in vitro to replicate within monocytes and macrophages and exit via non-lytic exocytosis [21][22][23][24]. Thus, it has been proposed that these cells can function as "Trojan horses" that facilitate fungal proliferation and dissemination [25][26][27]. Together, these data support an alternative model in which IM could be detrimental in the host response to acute C. neoformans infection.
In this study, we sought to clarify the role of IM and their derivatives in a murine model of acute cryptococcosis using a highly virulent serotype A strain of C. neoformans. We utilized CCR2-DTR depleter mice [5] and a new constitutive CCR2-Cre mouse model to probe the functional role of IM in C. neoformans control and host survival. Interestingly, we found that in the absence of IM, murine survival is improved and there is decreased fungal burden in the lungs and disseminated sites, indicating that IM are harmful for host anti-cryptococcal immunity. We did not find experimental evidence that immunopathology or cellular crosstalk between IM and lymphocytes or eosinophils influence these infectious outcomes. However, we observed that IM in the lungs exhibit an alternatively activated (M2)-like macrophage transcriptional profile in response to C. neoformans. In particular, IM significantly upregulated expression of the gene encoding arginase 1 (ARG1), an M2 marker that may modulate immune responses due to its competition for L-arginine substrate with NOS2, a marker for classically activated (M1) macrophages [28][29][30]. M2 macrophages have previously been demonstrated to have decreased anti-cryptococcal activity against C. neoformans in vitro compared to classically activated (M1) macrophages [31]. Interestingly, the conditional knockout of Arg1 in IM and the deletion of STAT6, a transcriptional regulator of Arg1 [32,33], in hematopoietic cells could not reverse the impact of IM on host outcomes during acute cryptococcosis. In summary, our study defines a novel cell-intrinsic role for IM as mediators of detrimental host immune responses to a respiratory fungal pathogen and indicates that the subversion of these potential antifungal effector cells by C. neoformans occurs early in the IM response.

IM recruited to the lungs worsen infectious outcomes in an acute respiratory infection model of murine cryptococcosis
Our studies utilized an acute infection model in which C57BL/6 mice were administered 10 3 yeast cells of C. neoformans serotype A strain H99 intratracheally (i.t.). This infection model was uniformly fatal with a range of inocula from 10 2 to 10 5 yeast cells (S1A Fig). We found that IM accumulated in the lungs of mice within the first week after respiratory challenge and persisted during the course of progressive infection (Fig 1A). There was also an increase in pulmonary CD11b + DCs and macrophages during infection (S1B Fig), consistent with the known developmental relationships between lung-infiltrating IM and these immune cell subsets during fungal infections [5,12,13].
To determine the role of IM at the onset of infection, these cells were transiently ablated in CCR2-DTR mice [5] by administering intraperitoneal (i.p.) diphtheria toxin (DT) on days -1, +1 and +3 relative to infection with H99 ( Fig 1B). DT treatment of CCR2-DTR mice also resulted in significant decreases in DCs and macrophages in the lungs on day 7 post-infection (p.i.) (S2 Fig). DCs in CCR2-DTR mice returned in numbers comparable to non-transgenic littermate controls (WT) by day 14 p.i., while macrophages exhibited a slower return toward WT levels (S2 Fig). Compared to WT mice, CCR2-DTR mice had significantly prolonged survival (median survival 35.5 days for CCR2-DTR versus 27.5 days for WT) ( Fig 1C) and decreased fungal burden in the lungs on days 7, 14, 21 and 26 p.i. (Fig 1D). CCR2-DTR mice had a similar~1 log reduction in lung fungal burden compared to WT littermates when challenged with a 10-fold higher inoculum of 10 4 H99 yeast cells ( CCR2-DTR mice also had a decreased fungal burden in the mediastinal lymph node ( Fig 1E) and a trend toward a decreased fungal burden in the brain (Fig 1F) on days 21 and 26 p.i. compared to WT mice. These results suggest that IM and their derivatives are harmful to the host during acute cryptococcosis by promoting fungal proliferation and dissemination from the lungs.
To examine whether the recruitment of IM to the lungs promotes detrimental responses to C. neoformans, we utilized CCR2 -/mice [34], in which the ability of monocytes to migrate out of the bone marrow (BM) is significantly impaired, resulting in an~75% reduction in circulating monocytes under homeostatic conditions [35]. We confirmed that CCR2 -/mice have añ 79% decrease in the number of IM in the lungs compared to WT mice 14 days after C. neoformans challenge (Fig 2A). CCR2 -/mice demonstrated a significant survival benefit (median ) challenge with C. neoformans strain H99 relative to naive mice (dotted line). Data were pooled from eight independent experiments (n = 6-26 total mice per timepoint). (B) Diphtheria toxin (DT) was administered intraperitoneally (i.p.) as illustrated to ablate IM in CCR2-DTR mice. (C) Kaplan-Meier survival curve of WT littermate controls (white circles) and CCR2-DTR mice (black circles) after administration of DT and H99. Data were pooled from two independent experiments (n = 10 total mice per group). (D-F) CFU in (D) lung, (E) mediastinal lymph node (LN), and (F) brain homogenates from WT and CCR2-DTR mice at indicated timepoints. Data were pooled from nine independent experiments (n = 7-27 total mice per group per timepoint). � , P < 0.05. �� , P < 0.01. ��� , P < 0.001. ���� , P < 0.0001. https://doi.org/10.1371/journal.ppat.1007627.g001 survival 31.5 days for CCR2 -/versus 26 days for WT) ( Fig 2B) and a lower lung fungal burden on day 14 p.i. (Fig 2C) compared to WT mice. Collectively, these data indicate that the initial recruitment of IM to the lungs plays a key role in mediating harmful outcomes during acute cryptococcosis. Consistent with this model, IM depletion later in the course of infection, by administering DT on days +6, +8, and +10 p.i. to CCR2-DTR mice, did not give rise to any differences in survival between CCR2-DTR and WT mice (S3C Fig).

CCR2-Cre mice enable conditional knockout of genes in IM
To establish a strategy to investigate the role of IM functions in our model, we generated a CCR2-Cre mouse that allows us to conditionally knockout genes of interest in IM. We used a bacterial artificial chromosome (BAC) transgenic approach to introduce the Cre recombinase

IM do not regulate lymphocyte responses during acute cryptococcosis
Ablation of IM led to a reduction in the number of pulmonary lymphocytes, including natural killer (NK) cells, innate lymphoid cell subsets (ILCs), and CD4 + T cells on day 7 p.i. (Fig 4), suggesting that IM may regulate lymphocyte activity against C. neoformans. We also found a marked reduction in the levels of the Th2 cytokines IL-5 and RANTES/CCL5 along with a slight decrease in TNF production at the same timepoint in IM-ablated CCR2-DTR mice (S6 Fig). These data indicated that IM could be deleterious because they facilitate the generation of harmful Th2 responses during acute cryptococcosis. To examine the possible link between MHC class II (MHCII) antigen presentation by IM and host Th2 responses, we generated CCR2-Cre MHCII fl/fl mice to conditionally knockout MHCII expression in IM and their derivative cells. We confirmed the loss of MHCII expression in a subset of CD11b + CD11c + cells, consistent with IM-derived DCs (S7 Fig). However, conditional knockout of MHCII in these cells had no effect on survival ( Fig 5A) or lung fungal burden on day 14 p.i. (Fig 5B) compared to MHCII fl/fl littermate controls. Therefore, MHCII antigen presentation by IM and their derivatives does not mediate harmful immune responses to C. neoformans.
To determine if lymphocytes are essential for IM-mediated outcomes, we next crossed the CCR2-DTR mice with RAG -/γc -/mice, in which lymphocytes, including T-and B-cells, NK cells, and ILCs, are absent. These CCR2-DTR RAG -/γc -/mice enabled DT-mediated ablation of IM in a lymphocyte-deficient background. We found that CCR2-DTR RAG -/γc -/mice had improved survival compared to non-transgenic RAG -/γc -/littermates (median survival of 28 days for CCR2-DTR RAG -/γc -/versus 23 days for RAG -/γc -/-) ( Fig 6A) and a decreased lung fungal burden on day 7 p.i. (Fig 6B). Therefore, lymphocytes were not required to mediate the detrimental effects of IM in our model. We note that although IM are the most prevalent CCR2-expressing cells, subsets of T cells, NK cells, and ILC precursors can express variable levels of CCR2 [4,37,38]. Thus, these results confirm that IM, not other CCR2-expressing lymphocytes, were responsible for the phenotypes we observed.

Eosinophils do not regulate host outcomes in acute cryptococcosis
Previous studies have suggested that eosinophils are associated with cryptococcal disease in humans and mice [39-48] and positively correlate with murine susceptibility to cryptococcosis [45,49]. We observed a reduction in eosinophils on days 7 and 14 p.i. in the lungs of IM-ablated CCR2-DTR mice compared to WT littermates (Fig 4). Since the eosinophil-active cytokines IL-5 and RANTES/CCL5 were also diminished in CCR2-DTR mice (S6 Fig), we investigated whether eosinophils may mediate the downstream effects of IM. After respiratory challenge with C. neoformans, eosinophil-deficient ΔdblGATA mice [50] did not exhibit any differences in survival (Fig 7A) or lung fungal burden on days 7 and 14 p.i. (Fig 7B) compared to WT control mice. These findings indicate that pulmonary eosinophilia likely represents a byproduct of an ineffective, Th2-skewed immune response rather than a functional immune response to C. neoformans. Therefore, eosinophils are unlikely to mediate the harmful effects of IM.

IM express M2 macrophage markers in response to respiratory C. neoformans challenge
Since our data suggested that cellular crosstalk between IM and lymphocytes or eosinophils is not a primary mechanism by which IM regulate infectious outcomes, we next examined the role of direct intrinsic functions of IM in mediating detrimental immune responses to C. neoformans. RNASeq analysis of IM sorted from the lungs of naive and infected CCR2-GFP reporter mice [5] on days 5 and 10 p.i. was performed ( Fig 8A). We found that, compared to naive IM, infected IM demonstrated significant increases (P adj < 0.05) in the transcription of genes commonly associated with an alternatively activated (M2) macrophage phenotype [51], including Arg1, mannose receptor C-type 1 (Mrc1/Cd206), transglutaminase 2 (Tgm2), resistin like alpha/found in inflammatory zone 1 (Retnla/Fizz1), and the chemokines Ccl17 and Ccl24. Compared to naive IM, infected IM on day 10 p.i. also exhibited a high suppressor of cytokine signaling (Socs)1:Soc3 ratio, that has been associated with the M2 phenotype [52, 53]. There was no differential expression of the M2 marker chitinase-like 3 (Chil3/Ym1). Among transcripts associated with classically activated (M1) macrophages, Nos2 was not detected by RNA-Seq, and, except for a slight increase in the chemokine Cxcl9 on day 10 p.i. (P adj < 0.05), there was no differential expression of the remaining transcripts. Among DC-associated transcripts, there was a slight increase in Jak2 on day 10 p.i. (P adj < 0.05) but no changes in other transcripts, including those of the zinc finger and BTB domain containing 46 transcription factor (Zbtb46) and MHC class II molecules (H2-Eb1, H2-Eb2, H2-DMa, H2-Ab1).
The RNASeq findings were validated by qRT-PCR in naive and infected mice on day 10 p.i. (Fig 8B). We confirmed there was no significant change in the M1 marker Nos2, but we did detect a slight decrease in expression of Tnf in infected IM. We again saw significant increases in the M2 markers Arg1, Mrc1 and Retnla/Fizz1 and no change in Chil3/Ym1 transcription, but we also detected an increase in the V-maf musculoaponeurotic fibrosarcoma oncogene homolog B transcription factor (Mafb), that promotes macrophage differentiation [54]. Finally, we confirmed no change in the DC marker Zbtb46. Together, these results suggest that C. neoformans subverts IM to assume an M2 macrophage-like phenotype that may be more permissive for fungal proliferation.

Disruption of M2 polarization pathways does not reverse the harmful effects of IM
Arg1, an archetypal M2 macrophage marker, was one of the most highly expressed transcripts in infected IM (Fig 8). Previous studies indicate that ARG1 may compete with the M1 macrophage marker NOS2 for L-arginine substrate that it metabolizes into urea and L-ornithine, thereby reducing nitric oxide production by NOS2 [28-30]. Since M2 macrophages are less fungicidal than M1 macrophages against C. neoformans in vitro [31], we investigated the potential role of IM-intrinsic ARG1 activity in the response to C. neoformans in the lungs by generating CCR2-Cre Arg1 fl/fl mice. Our results showed that conditional knockout of Arg1 in IM decreased total arginase activity in the lungs by approximately 50% (S8A Fig) but did not have any effect on survival ( Fig 9A) or lung fungal burden on days 7 and 14 p.i. (Fig 9B). Additionally, we observed that Vav1-Cre Arg1 fl/fl mice, that lack Arg1 in all hematopoietic cells, had no difference in survival compared to Arg1 fl/fl or fl/+ littermate controls (S8B Fig). Therefore, although infected IM highly express Arg1, it does not appear that Arg1 mediates the detrimental effects of IM or other hematopoietic cells in response to C. neoformans challenge.
STAT6 is a transcription factor that mediates M2 macrophage polarization in the context of IL-4 and IL-13 signaling by regulating expression of M2 macrophage markers, including Arg1, Mrc1, and Retnla/Fizz1 [55,56]. To determine if more global blockade of M2 macrophage polarization pathways would reverse the detrimental host outcomes in our model of cryptococcosis, we generated STAT6 -/-BM chimeras to knockout STAT6 in hematopoietic cells, since STAT6 fl/fl mice are not commercially available. There was no difference in overall survival ( Fig 9C) and lung fungal burden on days 7 and 14 p.i. (Fig 9D) in STAT6 -/-BM chimeras compared to control mice. Therefore, blocking STAT6-mediated pathways in hematopoietic cells, including IM, does not appear to be sufficient to counteract the harmful effects of IM during acute cryptococcosis. These results suggest that STAT6-regulated M2 markers, while associated with this macrophage phenotype, do not play an active role in macrophage function during acute cryptococcosis and that the subversion of IM function by C. neoformans more likely occurs at an earlier stage in the host-pathogen interaction.

Discussion
In this study, we establish a critical role for IM in mediating detrimental host outcomes in a model of fatal respiratory infection with C. neoformans. These findings contrast with the beneficial functions of IM described in murine models of subacute and chronic pulmonary cryptococcosis and other fungal infections [4-8, 11, 14, 15] but align with studies that suggest IM and their derivatives are associated with progression of infection by C. neoformans [20][21][22][23][24][25][26][27]. The studies on IM during subacute and chronic cryptococcosis utilized CCR2 -/mice on a BALB/c or mixed C57BL/6 and 129 background with the less virulent C. neoformans serotype D strain 52D [11,14,15]. We observed an improvement in lung fungal burden with ablation of IM in CCR2-DTR mice on the C57BL/6 background infected with 52D. However, Murdock et al. have shown that infection of C57BL/6 mice with the same inoculum of 52D i.t. is nonfatal up to 8 weeks p.i., suggesting this combination represents a more chronic model of infection [57]. Therefore, mouse genetic background may be an important factor in determining the role of IM in the immune response to C. neoformans, aside from the acuity of the infection, though we cannot yet exclude any contribution from differences in the fungal strains themselves based on available data. In any case, these disparate results indicate that IM possess a plasticity of function that can regulate the outcomes of infection and, thus, would make them an important target for immunomodulatory therapies against C. neoformans.
Accordingly, we sought to identify the mechanisms by which IM regulate infectious outcomes during cryptococcosis. To aid our investigation, we generated a CCR2-Cre transgenic mouse that demonstrates excellent Cre activity in IM, especially in the lungs. Along with the inducible Ccr2-creER T2 mouse previously generated by Becker and colleagues [58], the constitutive CCR2-Cre mouse expands the tools available to dissect IM function in a variety of immunologic processes.
One of our first observations was that IM contribute to Th2 cytokine production and regulate the presence of NK cells, ILCs, and CD4 + T cells in the lungs after C. neoformans challenge. IM and their macrophage and DC derivatives often play important roles in regulating lymphocyte responses to pulmonary infections through antigen presentation and chemokine secretion https://doi.org/10.1371/journal.ppat.1007627.g009 [5,59,60]. Interestingly, we found that neither conditional deletion of MHCII in IM nor lymphocyte deficiency affected IM-mediated infectious outcomes, suggesting that IM-lymphocyte crosstalk is not relevant for host immune responses during acute C. neoformans infection. The cellular source of IL-5 and RANTES/CCL5 in our model remains unclear. We did detect some transcription of RANTES/CCL5, but not IL-5, by IM in our RNASeq data. RANTES/CCL5 has been reported to be expressed by many cell types (immgen.org [61]) while the sources of IL-5 are typically Th2 cells and type 2 ILCs [62-64]. Thus, although the observed changes in these cytokines upon IM ablation may be related to direct secretion by IM, an alternative possibility is that differences in fungal burden influenced cytokine secretion by other immune cells, or a combination thereof.
We also investigated whether IM regulate the immune response to C. neoformans through the recruitment of eosinophils to the lungs. Some studies have suggested that eosinophils may play beneficial roles in rat models of cryptococcosis [46, 65, 66] or other fungal infections like aspergillosis [67][68][69]. However, eosinophilia has been correlated with poor outcomes in mouse and human cryptococcosis [39][40][41][42][43][44][45][46][47][48][49], and in the presence of IM in our model, we observed an upregulation of pulmonary IL-5 and RANTES/CCL5, cytokines that play important roles in eosinophil maturation and trafficking [70]. Ultimately, we found that eosinophil-deficient ΔdblGATA mice have no change in infectious outcomes compared to WT mice. It has also been previously reported that IL-5 -/and eosinophil-deficient PHIL mice have little to no change in infectious outcomes during cryptococcosis, although the data was not formally shown [49]. Thus, our results confirm that eosinophils are a hallmark of the progression of cryptococcal infection, but do not appear, in and of themselves, to play an active role in mediating the outcomes of infection.
Since cellular crosstalk between IM and lymphocytes or eosinophils did not seem to play an important role in our model, we next investigated the intrinsic functions of IM that may facilitate progression of C. neoformans infection. Based on our transcriptional profiling of infected IM, it appears that these cells preferentially express markers of M2 macrophages in response to pulmonary challenge with C. neoformans. M2 macrophages are generally anti-inflammatory cells and are involved in tissue homeostasis or repair, though they can be phenotypically heterogenous [71]. It has previously been reported that C. neoformans may use monocytes and macrophages as protected reservoirs or "Trojan horses" that aid in fungal dissemination [21][22][23][24][25]27] and that M2 macrophages are less fungicidal against C. neoformans than M1 macrophages [31]. Additionally, our histopathology analysis demonstrated that the lack of IM decreases the number of macrophages and multinucleated giant cells in the lung parenchyma and increases the incidence of extracellular fungal organisms in the lungs. Therefore, these results support the idea that exposure to C. neoformans renders IM and their macrophage derivatives permissive for fungal proliferation, so that their physical absence from the lungs can actually ameliorate infectious outcomes. It remains unclear whether IM directly facilitate dissemination of C. neoformans in this acute infection model or whether the higher lymph node and brain fungal burdens in WT mice are simply a reflection of the higher lung fungal burden in these mice compared to IM-ablated CCR2-DTR mice. However, studies on other fungal and bacterial infections have previously demonstrated the ability of IM to participate in direct transport of microbes from the lung to disseminated sites [5,72,73].
Next, we examined if the harmful effects of IM during acute cryptococcosis could be reversed by targeting M2 macrophage polarization pathways. Arg1 is a known M2 marker that was markedly upregulated in infected IM. Previous studies indicate that the fungal pathogen C. albicans can suppress NOS2 activity and induce ARG1 activity in human macrophages, resulting in decreased NO production and improved fungal survival [74][75][76][77]. Our studies show that conditional knockout of Arg1 in IM does not improve survival or lung fungal burden after C. neoformans challenge. Although it has been suggested that ARG1 and NOS2 can compete for the same pool of L-arginine substrate [29,30,78,79], it may be that disrupting ARG1 activity and increasing L-arginine availability is not sufficient. A second, positive signal may also be required to induce NOS2 transcription and activity, although it would be important to avoid triggering an uncontrolled inflammatory reaction that could lead to harmful immunopathology. Other studies have also indicated that despite the association of Arg1 with M2 macrophages, ARG1 may not always be a functional component of the immune response [80].
We subsequently considered whether the functional outcomes of M2 polarization may be controlled further upstream. Previous studies indicate that global deletion of IL-13, IL-4, and IL-4Rα, as well as conditional knockout of IL-4Rα in myeloid cells, improves murine outcomes after respiratory challenge with C. neoformans [81][82][83]. Therefore, we investigated the role of STAT6, a transcription factor stimulated by IL-4 and IL-13 that regulates the expression of several M2 markers including Arg1 [55, 56]. Knockout of STAT6 in hematopoietic cells using BM chimeras resulted in similar survival and lung fungal burden as control mice. These results contrast with the worse survival of global STAT6 -/mice infected with C. neoformans strain KN99α, an H99-derived strain, observed by Wiesner et al [64]. Since we used BM chimeras, it is possible that a radioresistant cell population is contributing to the phenotype observed by Wiesner et al or that differences between the parental H99 strain and KN99α could be in play. Nevertheless, our results indicate that STAT6 signaling in IM does not play an important role in our infection model and suggest that C. neoformans may suppress proinflammatory signals further upstream that would otherwise direct the differentiation of IM into fungicidal M1 macrophages. For example, we previously observed that mice deficient in the DAP12 signaling adapter have improved infectious outcomes after C. neoformans challenge and that DAP12-deficient macrophages have improved uptake and killing of C. neoformans in vitro [84]. Thus, DAP12-mediated pathways may be important targets for promoting a more beneficial, classically activated immune response to C. neoformans.
Additional unanswered questions about the role of IM during cryptococcosis remain. We do not yet know if IM may influence the function of other myeloid cells like neutrophils, the role of which remains unclear during C. neoformans infection (reviewed in [85]), or of nonmyeloid cells, e.g., lung epithelial cells, that can coordinate innate immune responses to other fungal infections [86]. Given the potential influence of mouse genetic background and fungal strain on IM function, it may be important to evaluate not only host immune differences, but also fungal-specific factors like capsule composition and Titan cell formation that may alter host-pathogen interactions [87,88].
In summary, our study establishes a novel role for IM as crucial arbiters of infectious outcomes during acute cryptococcosis. Unlike in other pulmonary and disseminated fungal infections [4][5][6][7][8], IM do not aid in host defense but rather are subverted by C. neoformans to maintain a passive state that can be harnessed by the fungus for replication and dissemination. Our work was assisted by the generation of a CCR2-Cre mouse that will facilitate continued mechanistic evaluation of IM function in cryptococcosis. Using genetic reprogramming to target pathways that result in classical activation of IM and aid in fungal clearance would validate the concept that immunomodulation can be developed as a new therapeutic approach to manage cryptococcal infections.

Chemicals and reagents
Chemicals were from Sigma-Aldrich, cell culture reagents were from Life Technologies/Gibco, and microbiological culture media were from BD Biosciences unless otherwise noted. Arginase activity was measured using an Arginase Activity Assay Kit (Sigma). Antibodies for flow cytometry were purchased from BD Biosciences, eBioscience or Tonbo unless otherwise indicated. Restriction enzymes were from New England Biolabs.

Ethics statement
All animal studies were performed with approval from the MSKCC Institutional Animal Care and Use Committee under protocol 13-07-008 and were compliant with all applicable provisions established by the Animal Welfare Act and the Public Health Services (PHS) Policy on the Humane Care and Use of Laboratory Animals.

Generation of the CCR2-Cre mouse
A bacterial artificial chromosome (BAC) containing the CCR2-Cre transgene was generated using the recombineering strategy developed by Heintz and colleagues [96,97]. Briefly, the BAC clone RP23-182D4 containing the endogenous CCR2 locus [5] was obtained from the BACPAC Resource Center at the Children's Hospital Oakland Research Institute (CHORI). A 970 bp fragment upstream and a 711 bp fragment downstream of the CCR2 start codon were amplified (see all primers in Table 1). The 987 bp Cre gene was amplified from the CreER t2 frt Kan(R) frt plasmid [98], kindly provided by Dr. T. Buch (Univ. of Zurich). Overlap PCR was performed with these three fragments to generate a 2668 bp CCR2-Cre recombination cassette, in which the Cre gene is flanked by two homology boxes from the CCR2 gene and the first nucleotide of the CCR2 endogenous locus after the Cre stop codon is deleted (Fig 3A). The recombination cassette was cloned into the AscI and NotI sites of the pLD53.SC-AB shuttle vector [96], previously provided by Dr. D. Littman (NYU) [5]. The cassette was sequenced with overlapping primer sets to ensure the absence of mutations. For homologous recombination, the modified shuttle vector was electroporated into the BAC clone RP23-182D4. Clones that underwent BAC cointegration and resolution were isolated by chloramphenicol and ampicillin selection followed by sucrose negative selection, as previously described [5,96]. Proper integration of the CCR2-Cre construct into the BAC was confirmed by Southern blot (S5A Fig) and sequencing of the modified regions. The CCR2-Cre BAC was then purified and injected into fertilized C57BL/6J oocytes by the University of Michigan Transgenic Animal Core. Four potential founder mice were identified out of 30 pups screened by PCR. These founders were bred to Rosa26 flSTOP-tdRFP mice and immune cells in the BM, blood, lungs and spleen were evaluated by flow cytometry. The progeny of two founders exhibited comparable tdRFP expression in a high percentage of monocytes (Fig 3B), and the lineage of one of these founders (#584) was chosen to establish the CCR2-Cre colony.

Infection with Cryptococcus neoformans
C. neoformans serotype A strain H99 #4413 was kindly provided by Dr J. Heitman (Duke). C. neoformans serotype D strain 52D (24067) was obtained from ATCC. All C. neoformans strains were maintained and grown as previously described [84]. Briefly, fungal strains were grown on Sabouraud dextrose agar (SAB) plates from frozen glycerol stocks and then cultured overnight at 37˚C in liquid YPD medium (1% yeast extract, 2% peptone, 2% dextrose). Fungal cells were then washed and resuspended in phosphate-buffered saline (PBS) at a concentration of 10 3 cells per 50 μl volume. Mice were anesthesized with inhaled isoflurane and given 50 μl of the fungal cell suspension intratracheally using a blunt-ended 20-gauge needle, as previously described [100].

Analysis of infected mice
To assess fungal burden, murine lung and brain tissue were mechanically homogenized in PBS using a PowerGen 125 homogenizer (Fisher). Lymph nodes were dissociated using ground glass slides. CFU in all tissues were counted after plating serial dilutions of the homogenates on SAB plates.

Histopathology
The lungs of euthanized mice were perfused with 4% paraformaldehyde (PFA) in situ via a catheter inserted through an incision in the trachea. The lungs were then harvested and fixed by immersion in 4% PFA. Lungs were then processed by the MSKCC Molecular Cytology Core Facility to generate 4 μm sections of paraffin-embedded lungs stained with hematoxylin & eosin (H&E). Slides were scanned using a Zeiss Mirax Midi slide scanner with 20x/0.8NA objective. Slides were reviewed and scored by a pathologist. Morphometry analysis was carried out on scanned slide images using Pannoramic Viewer (v1.15.3, 3DHISTECH). Areas of lung inflammation were measured and expressed as a percentage of total lung area in each histologic section.
RNASeq was performed by the MSKCC Integrated Genomics Operation. After RiboGreen quantification and quality control by an Agilent BioAnalyzer, 500ng of total RNA underwent polyA selection and TruSeq library preparation according to instructions provided by Illumina (TruSeq Stranded mRNA LT Kit, catalog # RS-122-2102), with 8 cycles of PCR. Samples were barcoded and run on a HiSeq 2500 in a 50bp/50bp paired end run, using the TruSeq SBS Kit v4 (Illumina). An average of 44.6 million paired reads was generated per sample. At the most the ribosomal reads represented 0.01% of the total reads generated and the percent of mRNA bases averaged 73.5%. The RNASeq data have been deposited in NCBI's Gene Expression Omnibus (GEO) [101] and are accessible through GEO Series accession number GSE122765 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE122765).
Statistical analysis of RNASeq data was performed by the MSKCC Bioinformatics Core. The output data (FASTQ files) were mapped to the target genome using the rnaStar aligner [102] that maps reads genomically and resolves reads across splice junctions. The 2 pass mapping method [103] was used, in which the first mapping pass uses a list of known annotated junctions from Ensembl. Novel junctions found in the first pass were then added to the known junctions and a second mapping pass was performed in which the RemoveNoncanoncial flag was used. After mapping, the output SAM files were post processed using the PICARD tool AddOrReplaceReadGroups to add read groups, sort the files, and covert them to the compressed BAM format. The expression count matrix was then computed from the mapped reads using HTSeq (www-huber.embl.de/users/anders/HTSeq) and one of several possible gene model databases. The raw count matrix generated by HTSeq was then processed using the R/Bioconductor package DESeq (www-huber.embl.de/users/anders/DESeq) which was used to both normalize the full dataset and analyze differential expression between sample groups.
For quantitative RT-PCR, cDNA was generated from RNA using a QuantiTect Reverse Transcription Kit (Qiagen), and qRT-PCR was performed on a StepOnePlus Real Time PCR System (Applied Biosystems) using TaqMan

Statistical analysis
All results are expressed as mean ± SEM. A Mann-Whitney U test was used for statistical analysis of two group comparisons, and one-way ANOVA was used for three groups or more, unless otherwise noted. Survival data was analyzed by Mantel-Cox test. All statistical analyses were performed with GraphPad Prism software, v6.0f. A P value < 0.05 was considered significant and indicated with an asterisk.