CCR2 Mediates Conventional Dendritic Cell Recruitment and the Formation of Bronchovascular Mononuclear Cell Infiltrates in the Lungs of Mice Infected withCryptococcus neoformans

Abstract

Pulmonary clearance of the encapsulated yeast Cryptococcus neoformans requires the development of T1-type immunity. CCR2-deficient mice infected with C. neoformans develop a non-protective T2 immune response and persistent infection. The mechanisms responsible for this aberrant response are unknown. The objective of this study was to define the number, phenotype, and microanatomic location of dendritic cells (DC) residing within the lung of CCR2+/+ or CCR2−/− mice throughout a time course following infection with C. neoformans. Results demonstrate the CCR2-mediated recruitment of conventional DC expressing modest amounts of costimulatory molecules. DC recruitment was preceded by the up-regulation in the lung of the CCR2 ligands CCL2 and CCL7. Colocalization of numerous DC and CD4+ T cells within bronchovascular infiltrates coincided with increased expression of IL-12 and IFN-γ. By contrast, in the absence of CCR2, DC recruitment was markedly impaired, bronchovascular infiltrates were diminished, and mice developed features of T2 responses, including bronchovascular collagen deposition and IL-4 production. Our results demonstrate that CCR2 is required for the recruitment of large numbers of conventional DC to bronchovascular infiltrates in mice mounting a T1 immune response against a fungal pathogen. These findings shed new insight into the mechanism(s) by which DC recruitment alters T cell polarization in response to an infectious challenge within the lung.

Chemokine Receptor 2-Mediated Accumulation of Fungicidal Exudate Macrophages in Mice That Clear Cryptococcal Lung Infection

“…In uninfected mice (D0), there were no differences between CCR2 ϩ/ϩ (wild-type) mice and CCR2 Ϫ/Ϫ mice in either AM or ExM populations ( Figure 7, A and B). Consistent with prior studies, 31,32,41 infection of CCR2 Ϫ/Ϫ mice with C. neoformans resulted in diminished accumulation of lung leukocytes (data not shown). However, AMs were not significantly reduced in CCR2 Ϫ/Ϫ mice (relative to CCR2 ϩ/ϩ mice) at any point after infection ( Figure 7A).…”

“…As expected, 31,32,41,45 infection stimulated a substantial accumulation (22-fold relative to D0) of lung leukocytes, peaking at 14 dpi (1.1 Ϯ 0.2 ϫ 10 8 CD45 ϩ leukocytes per lung). AMs were more numerous than ExMs in uninfected mice ( Figure 1E) but demonstrated only a modest fivefold increase relative to D0, peaking at 21 dpi (3.5% Ϯ 1.0% of total CD45 ϩ leukocytes and 3.6 Ϯ 0.5 ϫ 10 6 AMs/lung).…”

“…Initial gates eliminated debris, red cells, and cell clusters and selected for CD45 ϩ leukocytes. T cells, B cells, and eosinophils were excluded 41,51 (data not shown). Next, we set consecutive gates which identified CD11c ϩ cells ( Figure 1A, left dot plot) and distinguished autofluorescent (AF ϩ ) macrophages from non-AF (AF Ϫ ) dendritic cells (DCs) ( Figure 1A, middle dot plot), as previously described.…”

“…Lungs were then excised, minced, and enzymatically digested to obtain a single-cell suspension of lung leukocytes. 41 The peripheral blood mononuclear cells were isolated from peripheral blood obtained from the retro-orbital vein of deeply anesthetized mice and processed as previously described. 42 Spleens were excised and leukocytes were dispersed by mincing and mechanical disruption.…”

“…Next, we set consecutive gates which identified CD11c ϩ cells ( Figure 1A, left dot plot) and distinguished autofluorescent (AF ϩ ) macrophages from non-AF (AF Ϫ ) dendritic cells (DCs) ( Figure 1A, middle dot plot), as previously described. 41,45,[51][52][53] Last, we used relative expression of CD11b to distinguish CD11b-negative AMs (CD11c ϩ AF ϩ CD11b Ϫ ) 42,54,55 and CD11b-positive ExMs (CD11c ϩ AF ϩ CD11b ϩ ) 33,35 ( Figure 1A, right dot plot; gates labeled AM and ExM). Proper gate placement for AF and expression of CD11c and CD11b were determined using AMs obtained from bronchoalveolar lavage fluid of uninfected mice (which consists of Ͼ90% CD11c ϩ AF ϩ CD11b Ϫ AMs; data not shown).…”