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

Abstract

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.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Fig 1. Inflammatory monocytes promote detrimental host responses to Cryptococcus neoformans.

(A) IM in the lungs of C57BL/6 (WT) mice after intratracheal (i.t.) 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.

Fig 2. Trafficking of inflammatory monocytes to the lungs regulates detrimental immune responses.

(A) IM in the lungs of WT mice (white circles) and CCR2-/- mice (gray circles) on day 14 after i.t. challenge with H99. Data are from one experiment (n = 6 mice per group). (B) Kaplan-Meier survival curve of WT and CCR2-/- mice challenged with H99. Data were pooled from two independent experiments (n = 10–12 total mice per group). (C) CFU in the lungs of WT and CCR2-/- mice on day 14 p.i. Data are from one experiment (n = 6 mice per group). **, P < 0.01.

Fig 3. Generation and validation of a CCR2-Cre mouse.

(A) Schematic of the modification of a BAC containing the endogenous CCR2 locus in order to insert the Cre recombinase gene, a stop codon, and a frameshift mutation downstream of the CCR2 promoter. (B) Representative flow plots and histograms of tdRFP expression by IM in the bone marrow, blood and lung of naive CCR2-Cre Rosa26flSTOP-tdRFP mice. The dotted lines in the histograms represent naive Rosa26flSTOP-tdRFP control mice.

Fig 4. Inflammatory monocytes regulate the presence of other immune cells in the lungs.

Natural killer cells, innate lymphoid cells, CD4+ T cells, and eosinophils in the lungs of WT mice (white circles) and CCR2-DTR mice (black circles) challenged with H99 were ennumerated on days 7 and 14 p.i. Data were pooled from four independent experiments (n = 5–14 total mice per group). **, P < 0.01. ****, P < 0.0001.

Fig 5. Inflammatory monocytes regulate detrimental immune responses to C. neoformans independent of MHCII expression.

(A) Kaplan-Meier survival curve of MHCIIfl/fl control mice (white circles) and CCR2-Cre MHCIIfl/fl mice (black circles) challenged with H99. Data are from one experiment (n = 7–8 mice per group). (B) CFU in the lungs on day 14 p.i. Data are from one experiment (n = 4–6 mice per group).

Fig 6. Lymphocytes are not essential for the detrimental effects of inflammatory monocytes.

(A) Kaplan-Meier survival curve of RAG-/-γc-/- control mice (white triangles) and CCR2-DTR RAG-/-γc-/- mice (black triangles) challenged with H99. Data are from two independent experiments (n = 9–10 mice per group). (B) CFU in the lungs on day 7 p.i. Data are from two independent experiments (n = 9–11 mice per group). **, P < 0.01; ****, P < 0.0001.

Fig 7. Eosinophils do not regulate infectious outcomes after C. neoformans challenge.

(A) Kaplan-Meier survival curve of WT mice (white circles) or ΔdblGATA mice (black circles) challenged with H99. Data were pooled from two independent experiments (n = 12–13 total mice per group). (B) CFU in the lungs on days 7 and 14 p.i. Data were pooled from two independent experiments (n = 4 total mice per group).

Fig 8. Inflammatory monocytes upregulate M2 macrophage markers in response to C. neoformans.

(A) Heat maps of the expression of M1 macrophage, M2 macrophage, and dendritic cell (DC) markers in pulmonary IM on days 5 and 10 p.i. relative to IM from naive mice. X = not detected. Data are from one experiment (n = 6–7 mice per timepoint). (B) Quantitative RT-PCR of pulmonary IM from naive mice or mice on day 10 p.i. Data are from one experiment (n = 3 mice per group). **, P < 0.01 and *, P < 0.05 by t-test.

Fig 9. Blocking M2 macrophage polarization pathways does not reverse the detrimental effects of inflammatory monocytes.

(A) Kaplan-Meier survival curve of control Arg1flfl or fl/+ mice (white circles) and CCR2-Cre Arg1flfl mice (black circles). Data are from one experiment (n = 4–5 mice per group). (B) CFU in the lungs of Arg1flfl or fl/+ and CCR2-Cre Arg1flfl mice on days 7 and 14 p.i. Data are from two experiments (n = 4–5 mice per group). (C) Kaplan-Meier survival curve of control WT→ CD45.1+ bone marrow chimeras (white circles) and STAT6-/- → CD45.1+ bone marrow chimeras (gray circles). Data are pooled from two experiments (n = 8–9 total mice per group). (D) CFU in the lungs of WT→ CD45.1+ and STAT6-/-→ CD45.1+ bone marrow chimeras on days 7 and 14 p.i. Data are from two experiments (n = 4–5 mice per group).