In the present study we demonstrate that the chemokine receptor Ccr1 is associated with fatal neutrophil-mediated immunopathology selectively in the kidney in a mouse model of systemic Candida albicans infection. The receptor appears to act directly by promoting neutrophil trafficking from the blood into the kidney, but not until the late phase of the infection when it is turned on selectively by blood neutrophils and when most of its ligands Ccl3, Ccl5, Ccl6, Ccl7, Ccl8 and Ccl9 are expressed at high levels in the kidney. Our conclusions are based on detailed analysis of differences in clinical, pathological, microbiological, immunological and molecular parameters in the model between Ccr1+/+ and Ccr1−/− mice. Our study is the first identification of a specific chemoattractant receptor that mediates neutrophil trafficking into the kidney and drives neutrophil-mediated immunopathology and mortality in the mouse model of invasive candidiasis. Moreover, the conclusions regarding mechanism are based on a formal in vivo neutrophil trafficking study.
We focused on Ccr1 because phagocytes are known to be the main cellular mediators of the immune response in invasive candidiasis 
, and because our broad survey of the chemokine system in the model showed that phagocyte-targeted chemokines and their receptors, including Ccr1 and its ligands, were among the most highly up-regulated. Ccr1 is a chemokine receptor whose functional importance for neutrophils has been more apparent in mice than in humans 
, and it is also expressed on other hematopoietic and some non-hematopoietic cell types 
The outcome in any infectious disease is determined by a balance between host and pathogen factors. In our study, we found several lines of evidence supporting excessive neutrophil accumulation in the kidney resulting in neutrophil-mediated immunopathology as the underlying mechanism for Ccr1-mediated decreased survival in the model. First, Ccr1 deficiency did not affect tissue Candida
proliferation, an established correlate of survival in previous studies in the model of invasive candidiasis 
. Second, Ccr1 deficiency was associated with markedly decreased destructive inflammatory changes and tissue injury in the kidney; this was accompanied by less severe kidney failure in Ccr1−/−
mice, which is a major determinant of survival in the model 
. Third, the decreased kidney tissue injury was associated with a large and selective reduction in neutrophil accumulation in Ccr1−/−
kidneys after infection.
Neutrophils are key innate immune effector cells that play a critical role in phagocytosis and killing of Candida albicans
. In agreement, neutropenia is a major risk factor for mortality after invasive candidiasis in both mice and humans 
. Early neutrophil availability in particular has been shown to be critical for protection against invasive candidiasis in mice, as neutrophil depletion within the first 24 hours, but not at later time points post-infection, led to accelerated Candida
growth and mortality 
. On the other hand, neutrophils may exert detrimental effects on the host by mediating tissue injury. For example, patients with invasive candidiasis often require administration of corticosteroid therapy after recovery from neutropenia for amelioration of clinical symptoms and attenuation of exuberant inflammation 
. A similar immune reconstitution inflammatory syndrome has also been described following neutrophil recovery in patients with invasive aspergillosis 
. In mice, the pathogenic role of neutrophils in invasive candidiasis has been previously suggested by neutrophil depletion studies 
, in which rendering mice neutropenic at late time points after infection when inflammation is pronounced 
, markedly improved survival, presumably due to attenuated tissue injury. Our study is the first to identify a single molecular factor, Ccr1, as one of the mediators of immunopathology in the mouse model of invasive candidiasis.
Ccr1 has been shown to exert both pathogenic and protective effects depending on the inflammatory milieu. For example, Ccr1 was critical for effective host defense against Toxoplasma gondii
, pneumovirus of mice and Aspergillus fumigatus
by mediating neutrophil accumulation in the target infection organs 
. Conversely, Ccr1-mediated neutrophil accumulation was pathogenic in a model of acute respiratory distress syndrome following pancreatitis and a model of sepsis following bacterial peritonitis 
. The role of Ccr1 has also been studied in the context of renal inflammation. Specifically, Ccr1 deficiency decreased neutrophil and macrophage accumulation in the kidney after ischemia-reperfusion injury, but it had no effect on kidney function or survival 
. Moreover, Ccr1 deficiency decreased kidney injury in models of Alport disease, focal segmental glomerulosclerosis, unilateral ureteral obstruction and toxic nephropathy; however, the attenuated tissue damage was associated with decreased infiltration of lymphocytes and macrophages but not neutrophils in the kidney 
The mechanism of Ccr1-mediated neutrophil accumulation and immunopathology in the kidney in invasive candidiasis appears to lie at the level of neutrophil trafficking from the blood into the kidney, as shown by our competitive repopulation study using Ccr1+/+
neutrophils. Additional work using gene-deficient mice and/or antibody depleting strategies will be needed to define which of the Ccr1 ligands, either alone or in synergy, mediate neutrophil trafficking into Candida
-infected kidneys. Also, because Ccr1 is expressed on neutrophil precursors and mature neutrophils in the bone marrow 
, the impact of Ccr1 deficiency in neutrophil precursor production and egress of neutrophils from the bone marrow to the blood after Candida
infection merits further investigation. Of note, although Ccr1 is expressed on several immune cell types 
, the reduction in leukocyte accumulation in Ccr1−/−
kidneys was specific to neutrophils. In addition, neutrophil accumulation was Ccr1-dependent only in the kidney but not in the other organs examined, further attesting to the organ-specific nature of the innate immune response in invasive candidiasis 
Of interest, Ccr1 mediated neutrophil accumulation in the kidney only late in the course of invasive candidiasis. Three important new questions arise from this finding: the first relates to the signals responsible for Ccr1 induction on neutrophils late but not early in the course of invasive candidiasis. Factors that have been reported to induce Ccr1 on the surface of mouse and human neutrophils include TNF-α, GM-CSF and IFN-γ 
, which were not found to be different between days 3 and 9 in blood or kidney in our model (data not shown). Candida albicans
grows both in the yeast and hyphal forms in the kidney starting immediately post-infection 
, but unmasking of β-glucan, the pathogen-associated molecular pattern for dectin-1 
, does not occur until late in the course of invasive candidiasis 
. Hence, it is plausible that availability of soluble or membrane-bound β-glucan late after invasive candidiasis may contribute to Ccr1 induction on neutrophils.
The second important new question that arises from our study pertains to the chemotactic factors responsible for neutrophil recruitment in the kidney during the first 6 days after infection, when Ccr1 is dispensable for neutrophil accumulation in the kidney. Other neutrophil chemokine receptors such as Cxcr1 and Cxcr2, which were also induced in the model, may play such a role. In this regard, a sequential role for the leukotriene B4
receptor Blt1, Ccr1 and Cxcr2 has previously been reported for neutrophil recruitment in a mouse model of arthritis 
. Because neutrophil availability during the early phase after infection is critical for survival 
, identifying the chemoattractant receptors that mediate trafficking into the kidney early in the course of the infection may reveal novel protective molecular factors against invasive candidiasis in the model. In fact, Cxcr2-deficient mice were previously reported to have impaired ability to control Candida
proliferation in the kidney and other organs after invasive candidiasis, but accumulation of neutrophils in these tissues was not defined in that study 
The third important new question arises from our data that neutrophil accumulation was decreased but not entirely eliminated in Ccr1−/− kidneys late after infection (), and from our competitive repopulation study, which showed that, despite the significant skewing toward Ccr1+/+ neutrophils, Ccr1−/− neutrophils also trafficked from the blood into the kidney at day 9 post-infection (). Thus, future studies should aim to determine which chemotactic factors, in addition to Ccr1, also mediate neutrophil trafficking from the blood into the kidney late after infection. Identification of such factors may reveal other novel mediators of immunopathology in the model and could provide further insight into the balance between effective host defense and immunopathology in invasive candidiasis.
In contrast to the mouse model of the infection where kidney is the primary target organ 
, kidney abscesses are rare in patients with bloodstream-derived invasive candidiasis with the exception of neonates 
; hence, whether CCR1 mediates immunopathology in human invasive candidiasis remains to be elucidated. Therefore, future studies should examine whether CCR1 is expressed on human neutrophils in Candida
kidney abscesses. In addition, because neutropenic patients with gastrointestinal tract-derived hepatosplenic candidiasis often develop worsening liver and splenic abscesses immediately after neutrophil recovery 
, the role of Ccr1 in mediating immunopathology in that setting should be investigated using a neutropenic mouse model of gastrointestinal tract-derived invasive candidiasis 
, in which it would be important to determine whether neutrophils that repopulate the bloodstream after recovery from neutropenia express Ccr1.
In conclusion, our data identify Ccr1 as a critical pathogenic factor in systemic infection caused by Candida albicans in a mouse model. Our study shows for the first time that neutrophil-mediated immunopathology may be detrimental in an invasive fungal infection and that chemokine receptors regulate such a process. Further studies will be needed to investigate the role of Ccr1 ligands and to further understand the factors that shape and modulate protective versus pathogenic roles of neutrophils in invasive candidiasis.