The Ly-6Chigh monocyte subpopulation transports Listeria monocytogenes into the brain during systemic infection of mice

Mononuclear phagocytes can be used by intracellular pathogens to disseminate throughout the host. In the bloodstream these cells are generically referred to as monocytes. However, blood monocytes are a heterogeneous population, and the exact identity of the leukocyte(s) relevant for microbial spreading is not known. Experiments reported in this study used Listeria monocytogenes-infected mice to establish the phenotype of parasitized blood leukocytes and to test their role in systemic dissemination of intracellular bacteria. More than 90% of the blood leukocytes that were associated with bacteria were CD11b(+) mononuclear cells. Analysis of newly described monocyte subsets showed that most infected cells belonged to the Ly-6C(high) monocyte subset and that Ly-6C(high) and Ly-6C(neg-low) monocytes harbored similar numbers of bacteria per cell. Interestingly, systemic infection with wild-type or DeltaactA mutants of L. monocytogenes, both of which escape from phagosomes and replicate intracellularly, caused expansion of the Ly-6C(high) subset. In contrast, this was not evident after infection with Deltahly mutants, which neither escape phagosomes nor replicate intracellularly. Importantly, when CD11b(+) leukocytes were isolated from the brains of lethally infected mice, 88% of these cells were identified as Ly-6C(high) monocytes. Kinetic analysis showed a significant influx of Ly-6C(high) monocytes into the brain 2 days after systemic infection. This coincided with both bacterial invasion and up-regulation of brain macrophage chemoattractant protein-1 gene expression. These data indicate that the Ly-6C(high) monocyte subset transports L. monocytogenes into the brain and establish their role as Trojan horses in vivo.     

CD11b+Ly-6C(hi) suppressive monocytes in experimental autoimmune encephalomyelitis

Innate immune cells may regulate adaptive immunity by balancing different lineages of T cells and providing negative costimulation. In addition, CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been described in tumor, parasite infection, and severe trauma models. In this study, we observe that splenic CD11b(+) cells markedly increase after experimental autoimmune encephalomyelitis (EAE) immunization, and they suppress T cell proliferation in vitro. Although >80% of CD11b(+) cells express varying levels of Gr-1, only a small population of CD11b(+)Ly-6C(high) inflammatory monocytes (IMC) can efficiently suppress T cell proliferation and induce T cell apoptosis through the production of NO. IFN-gamma produced by activated T cells is essential to induce IMC suppressive function. EAE immunization increases the frequencies of IMC in the bone marrow, spleen, and blood, but not in the lymph nodes. At the peak of EAE, IMC represent approximately 30% of inflammatory cells in the CNS. IMC express F4/80 and CD93 but not CD31, suggesting that they are immature monocytes. Furthermore, IMC have the plasticity to up-regulate NO synthase 2 or arginase 1 expression upon different cytokine treatments. These findings indicate that CD11b(+)Ly-6C(high) IMC induced during EAE priming are powerful suppressors of activated T cells. Further understanding of suppressive monocytes in autoimmune disease models may have important clinical implications for human autoimmune diseases.     

Colonic eosinophilic inflammation in experimental colitis is mediated by Ly6C(high) CCR2(+) inflammatory monocyte/macrophage-derived CCL11

Recent genome-wide association studies of pediatric inflammatory bowel disease have implicated the 17q12 loci, which contains the eosinophil-specific chemokine gene CCL11, with early-onset inflammatory bowel disease susceptibility. In the current study, we employed a murine model of experimental colitis to define the molecular pathways that regulate CCL11 expression in the chronic intestinal inflammation and pathophysiology of experimental colitis. Bone marrow chimera experiments showed that hematopoietic cell-derived CCL11 is sufficient for CCL11-mediated colonic eosinophilic inflammation. We show that dextran sodium sulfate (DSS) treatment promotes the recruitment of F4/80(+)CD11b(+)CCR2(+)Ly6C(high) inflammatory monocytes into the colon. F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocytes express CCL11, and their recruitment positively correlated with colonic eosinophilic inflammation. Phenotypic analysis of purified Ly6C(high) intestinal inflammatory macrophages revealed that these cells express both M1- and M2-associated genes, including Il6, Ccl4, Cxcl2, Arg1, Chi3l3, Ccl11, and Il10, respectively. Attenuation of DSS-induced F4/80(+)CD11b(+)CCR2(+)Ly6C(high) monocyte recruitment to the colon in CCR2(-/-) mice was associated with decreased colonic CCL11 expression, eosinophilic inflammation, and DSS-induced histopathology. These studies identify a mechanism for DSS-induced colonic eosinophilia mediated by Ly6C(high)CCR2(+) inflammatory monocyte/macrophage-derived CCL11.     

Plasticity of Ly-6C(hi) myeloid cells in T cell regulation

CD11b(+)Ly-6C(hi) cells, including inflammatory monocytes (IMCs) and inflammatory dendritic cells (IDCs), are important in infectious, autoimmune, and tumor models. However, their role in T cell regulation is controversial. In this article, we show that T cell regulation by IMCs and IDCs is determined by their activation state and is plastic during an immune response. Nonactivated IMCs and IDCs function as APCs, but activated IMCs and IDCs suppress T cells through NO production. Suppressive IMCs are induced by IFN-γ, GM-CSF, TNF-α, and CD154 derived from activated T cells during their interaction. In experimental autoimmune encephalomyelitis, CD11b(+)Ly-6C(hi) cells in the CNS are increasingly activated from disease onset to peak and switch their function from Ag presentation to T cell suppression. Furthermore, transfer of activated IMCs or IDCs enhances T cell apoptosis in the CNS and suppresses experimental autoimmune encephalomyelitis. These data highlight the interplay between innate and adaptive immunity: immunization leads to the expansion of Ly-6C(hi) myeloid cells initially promoting T cell function. As T cells become highly activated in the target tissue, they induce activation and NO production in Ly-6C(hi) myeloid cells, which in turn suppress T cells and lead to the contraction of local immune response.     

Loss of TNF signaling facilitates the development of a novel Ly-6C(low) macrophage population permissive for Leishmania major infection

In the absence of TNF, the normally resistant C57BL/6 (B6.WT) strain develops a fatal, progressive form of leishmaniasis after infection with Leishmania major. It is not yet understood which TNF activity or the lack thereof is responsible for the dramatic progression of leishmaniasis in TNF-negative (B6.TNF(-/-)) mice. To elucidate the underlying mechanisms resulting in the fatal outcome of L. major infection in this gene-deficient mouse strain, we analyzed the monocytic component of the inflammatory infiltrate in the draining popliteal lymph node and the site of the infection using multicolor flow cytometry. The leukocytic infiltrate within the draining lymph node and footpad of B6.TNF(-/-) mice resembled that of B6.WT mice over the first 2 wk of cutaneous L. major infection. Thereafter, the B6.TNF(-/-) mice showed an increase of CD11c(+)Ly-6C(+)CCR2(+) monocytic dendritic cells within the popliteal lymph node in comparison with B6.WT mice. This increase of inflammatory dendritic cells was paired with the accumulation of a novel CD11b(+)Ly-6C(low)CCR2(low) population that was not present in B6.WT mice. This B6.TNF(-/-)- and B6.TNFR1(-/-)-specific cell population was CD115(+)Ly-6G(-)iNOS(-), not apoptotic, and harbored large numbers of parasites.     

Blood monocyte subsets differentially give rise to CD103+ and CD103- pulmonary dendritic cell populations

There are two major myeloid pulmonary dendritic cell (DC) populations: CD103+ DCs and CD11bhigh DCs. In this study, we investigated in detail the origins of both myeloid DC pools using multiple experimental approaches. We show that, in resting lung, Ly-6ChighCCR2high monocytes repopulated CD103+ DCs using a CCR2-dependent mechanism, and these DCs preferentially retained residual CCR2 in the lung, whereas, conversely, Ly-6ClowCCR2low monocytes repopulated CD11bhigh DCs. CX3CR1 was required to generate normal numbers of pulmonary CD11bhigh DCs, possibly because Ly-6Clow monocytes in the circulation, which normally express high levels of CX3CR1, failed to express bcl-2 and may have diminished survival in the circulation in the absence of CX3CR1. Overall, these data demonstrate that the two circulating subsets of monocytes give rise to distinct tissue DC populations.     

TNF-alpha-dependent regulation of acute pancreatitis severity by Ly-6C(hi) monocytes in mice

The roles of monocytes/macrophages and their mechanisms of action in the regulation of pancreatitis are poorly understood. To address these issues, we have employed genetically altered mouse strains that either express the human diphtheria toxin receptor (DTR) coupled to the CD11b promoter or have global deletion of TNF-α. Targeted, conditional depletion of monocytes/macrophages was achieved by administration of diphtheria toxin (DT) to CD11b-DTR mice. We show that in the absence of DT administration, pancreatitis is associated with an increase in pancreatic content of Ly-6C(hi) monocytes/macrophages but that this response is prevented by prior administration of DT to CD11b-DTR mice. DT administration also reduces pancreatic edema and acinar cell injury/necrosis in two dissimilar experimental models of acute pancreatitis (a secretagogue-induced model and a model elicited by retrograde pancreatic duct infusion of sodium taurocholate). In the secretagogue-elicited model, the DT-induced decrease in pancreatitis severity is reversed by adoptive transfer of purified Ly-6C(hi) monocytes harvested from non-DT-treated CD11b-DTR mice or by the transfer of purified Ly-6C(hi) monocytes harvested from TNF-α(+/+) donor mice, but it is not reversed by the transfer of Ly-6C(hi) monocytes harvested from TNF-α(-/-) donors. Our studies indicate that the Ly-6C(hi) monocyte subset regulates the severity of pancreatitis by promoting pancreatic edema and acinar cell injury/necrosis and that this phenomenon is dependent upon the expression of TNF-α by those cells. They suggest that therapies targeting Ly-6C(hi) monocytes and/or TNF-α expression by Ly-6C(hi) monocytes might prove beneficial in the prevention or treatment of acute pancreatitis.     

CCL9 is secreted by the follicle-associated epithelium and recruits dome region Peyer's patch CD11b+ dendritic cells

The follicle-associated epithelium (FAE) secretes chemokines important in the recruitment of various cell types including CCL20 (MIP-3alpha). CCL20 is chemotactic to the CD11b(+) dendritic cells (DCs) distributed in the subepithelial dome regions of the Peyer's patches, and mice deficient in the receptor for CCL20, CCR6, have been reported to be devoid of the CD11b(+) DCs in the dome regions. Here, we describe another chemokine specifically secreted from the FAE of mouse Peyer's patches, CCL9 (MIP-1gamma, CCF18, MRP-2). By in situ hybridization, we demonstrated that CCL9 mRNA was expressed by the FAE but not by the villus epithelium. At the protein level, CCL9 was detected on the FAE and on extracellular matrix structures within the dome regions of the Peyer's patches. By RT-PCR, we demonstrated that one of the putative receptors for CCL9, CCR1, was expressed by the Peyer's patch CD11b(+) DCs and in a chemotaxis assay, CD11b(+) DCs migrated toward CCL9. To compare the abilities of the chemokines CCL20 and CCL9 to recruit CD11b(+) DCs to the dome regions, we examined the in vivo distribution of these cells in CCR6-deficient, CCL9-blocked wild type, or CCL9-blocked CCR6-deficient mice. To our surprise, using a sensitive immunofluorescence analysis, we observed that CD11b(+) DCs were present in the dome regions of the CCR6-deficient mice. In contrast, Ab neutralization of CCL9 in vivo resulted in significant reduction of the CD11b(+) DC number in the subepithelial dome regions of Peyer's patches of both wild type and CCR6 -/- mice. Taken together, these results demonstrate an important role of CCL9 in CD11b(+) DC recruitment to the dome regions of mouse Peyer's patches.     

Ly-6I, a new member of the murine Ly-6 superfamily with a distinct pattern of expression

A new member of the mouse Ly-6SF, designated Ly-6I, has been isolated as a gene homologous to a segment of the Ly-6C gene. A single allelic difference in the mature protein sequence was identified, which is similar to other Ly-6SF members. Ly-6I mRNA has been detected in a wide range of tissues and cell lines, and a rabbit polyclonal Ab has been used to determine that Ly-6I protein is present at a low constitutive level on cell lines from several different lineages. In contrast to Ly-6C and Ly-6A/E, the Ly-6I gene is only weakly responsive to IFNs. Expression in vivo is most abundant on bone marrow populations and is coexpressed with Ly-6C on granulocytes and macrophages. However, Ly-6I is also expressed on immature B cell populations that do not express Ly-6C. Expression on mature B cells in spleen is uniformly low. Similarly, Ly-6I is expressed on TCRlow/int, but not TCRhigh, thymocytes. Ly-6I is re-expressed on Ly-6Chigh T cells in the periphery. Thus, Ly-6I may be a useful marker to define maturation stages of both T and B lymphocytes as well as subsets of monocytes and granulocytes.     

Distinct Upstream Role of Type I IFN Signaling in Hematopoietic Stem Cell-Derived and Epithelial Resident Cells for Concerted Recruitment of Ly-6Chi Monocytes and NK Cells via CCL2-CCL3 Cascade

Type I interferon (IFN-I)-dependent orchestrated mobilization of innate cells in inflamed tissues is believed to play a critical role in controlling replication and CNS-invasion of herpes simplex virus (HSV). However, the crucial regulators and cell populations that are affected by IFN-I to establish the early environment of innate cells in HSV-infected mucosal tissues are largely unknown. Here, we found that IFN-I signaling promoted the differentiation of CCL2-producing Ly-6C^hi monocytes and IFN-γ/granzyme B-producing NK cells, whereas deficiency of IFN-I signaling induced Ly-6C^lo monocytes producing CXCL1 and CXCL2. More interestingly, recruitment of Ly-6C^hi monocytes preceded that of NK cells with the levels peaked at 24 h post-infection in IFN-I–dependent manner, which was kinetically associated with the CCL2-CCL3 cascade response. Early Ly-6C^hi monocyte recruitment was governed by CCL2 produced from hematopoietic stem cell (HSC)-derived leukocytes, whereas NK cell recruitment predominantly depended on CC chemokines produced by resident epithelial cells. Also, IFN-I signaling in HSC-derived leukocytes appeared to suppress Ly-6G^hi neutrophil recruitment to ameliorate immunopathology. Finally, tissue resident CD11b^hiF4/80^hi macrophages and CD11c^hiEpCAM⁺ dendritic cells appeared to produce initial CCL2 for migration-based self-amplification of early infiltrated Ly-6C^hi monocytes upon stimulation by IFN-I produced from infected epithelial cells. Ultimately, these results decipher a detailed IFN-I–dependent pathway that establishes orchestrated mobilization of Ly-6C^hi monocytes and NK cells through CCL2-CCL3 cascade response of HSC-derived leukocytes and epithelium-resident cells. Therefore, this cascade response of resident–to-hematopoietic–to-resident cells that drives cytokine–to-chemokine–to-cytokine production to recruit orchestrated innate cells is critical for attenuation of HSV replication in inflamed tissues.     

CCR2 and CCR6, but not endothelial selectins, mediate the accumulation of immature dendritic cells within the lungs of mice in response to particulate antigen

Dendritic cells (DC) migrate from sites of inflammation to lymph nodes to initiate primary immune responses, but the molecular mechanisms by which DC are replenished in the lungs during ongoing pulmonary inflammation are unknown. To address this question, we analyzed the secondary pulmonary immune response of Ag-primed mice to intratracheal challenge with the particulate T cell-dependent Ag sheep erythrocytes (SRBC). We studied wild-type C57BL/6 mice and syngeneic gene-targeted mice lacking either both endothelial selectins (CD62E and CD62P), or the chemokine receptors CCR2 or CCR6. DC, defined as non-autofluorescent, MHC class II(+)CD11c(mod) cells, were detected in blood, enzyme-digested minced lung, and bronchoalveolar lavage fluid using flow cytometry and immunohistology. Compared with control mice, Ag challenge increased the frequency and absolute numbers of DC, peaking at day 1 in peripheral blood (6.5-fold increase in frequency), day 3 in lung mince (20-fold increase in total DC), and day 4 in bronchoalveolar lavage fluid (55-fold increase in total DC). Most lung DC expressed CD11c, CD11b, and low levels of MHC class II, CD40, CD80, and CD86, consistent with an immature myeloid phenotype. DC accumulation depended in part upon CCR2 and CCR6, but not endothelial selectins. Thus, during lung inflammation, immature myeloid DC from the bloodstream replace emigrating immature DC and transiently increase total intrapulmonary APC numbers. Early DC recruitment depends in part on CCR2 to traverse vascular endothelium, plus CCR6 to traverse alveolar epithelium. The recruitment of circulating immature DC represents a potential therapeutic step at which to modulate immunological lung diseases.     

Hematopoietic miR155 deficiency enhances atherosclerosis and decreases plaque stability in hyperlipidemic mice

microRNA-155 (miR155) is a central regulator of immune responses that is induced by inflammatory mediators. Although miR155 is considered to be a pro-inflammatory microRNA, in vitro reports show anti-inflammatory effects in lipid-loaded cells. In this study we examined the role of miR155 in atherosclerosis in vivo using bone marrow transplantation from miR155 deficient or wildtype mice to hyperlipidemic mice. Hematopoietic deficiency of miR155 enhanced atherosclerotic plaque development and decreased plaque stability, as evidenced by increased myeloid inflammatory cell recruitment to the plaque. The increased inflammatory state was mirrored by a decrease in circulating CD4(+)CD25(+)FoxP3(+) regulatory T cells, and an increase in granulocytes (CD11b(+)Ly6G(+)) in blood of miR155(-/-) transplanted mice. Moreover, we show for the first time a crucial role of miR155 in monocyte subset differentiation, since hematopoietic deficiency of miR155 increases the 'inflammatory' monocyte subset (CD11b(+)Ly6G(-)Ly6C(hi)) and reduces 'resident' monocytes (CD11b(+)Ly6G(-)Ly6C(low)) in the circulation. Furthermore, cytokine production by resident peritoneal macrophages of miR155(-/-) transplanted hyperlipidemic mice was skewed towards a more pro-inflammatory state since anti-inflammatory IL-10 production was reduced. In conclusion, in this hyperlipidemic mouse model miR155 acts as an anti-inflammatory, atheroprotective microRNA. Additionally, besides a known role in lymphoid cell development, we show a crucial role of miR155 in myeloid lineage differentiation.     

Expression of an unusual T cell receptor (TCR)-V beta repertoire by Ly-6C+ subpopulations of CD4+ and/or CD8+ thymocytes. Evidence for a developmental relationship between Ly-6C+ thymocytes and CD4-CD8-TCR-alpha beta+ thymocytes.

A novel thymocyte subpopulation expressing an unusual TCR repertoire was identified by high surface expression of the Ly-6C Ag. Ly-6C+ thymocytes were distributed among all four CD4/CD8 thymocyte subsets, and represented a readily identifiable subpopulation within each one. Ly-6C+ thymocytes express TCR-alpha beta, arise late in ontogeny, and appear in the CD4/CD8 developmental pathway after birth in a sequence that resembles that followed by conventional Ly-6C- cells during fetal ontogeny. Most interestingly, adult Ly-6C+ thymocytes express an unusual TCR-V beta repertoire that is identical to that expressed by CD4-CD8-TCR-alpha beta+ thymocytes in its overexpression of TCR-V beta 8 and in its expression of some potentially autoreactive TCR-V beta specificities. This unusual TCR-V beta repertoire was even expressed by Ly-6C+ thymocytes contained within the CD4+ CD8- 'single positive' thymocyte subset. Thus, expression of this unusual TCR-V beta repertoire is not limited to CD4-CD8-thymocytes, and is unlikely to be a consequence of their double negative phenotype. Rather, we think that Ly-6C+TCR-alpha beta+ thymocytes and CD4-CD8-TCR-alpha beta+ are developmentally interrelated, a conclusion supported by several lines of evidence including the selective failure of both Ly-6C+ and CD4-CD8-TCR-alpha beta+ thymocyte subsets to appear in TCR-beta transgenic mice. In contrast, peripheral Ly-6C+ T cells are developmentally distinct from Ly-6C+ thymocytes in that peripheral Ly-6C+ T cells expressed a conventional TCR-V beta repertoire and developed normally in TCR-beta transgenic mice in which Ly-6C+ thymocytes failed to arise. We conclude that: 1) expression of a skewed TCR-V beta repertoire is a characteristic of Ly-6C+TCR-alpha beta+ thymocytes as well as CD4-CD8-TCR-alpha beta+ thymocytes, and is not unique to thymocytes expressing neither CD4 nor CD8 accessory molecules; and 2) Ly-6C+ thymocytes are developmentally linked to CD4-CD8-TCR-alpha beta+ thymocytes, but not to Ly-6C+ peripheral T cells. We suggest that Ly-6C+TCR-alpha beta+ thymocytes are not the developmental precursors of Ly-6C+ peripheral T cells, but rather may be the developmental precursors of CD4-CD8-TCR-alpha beta+ thymocytes.     

Successful elimination of memory-type CD8+ T cell subsets by the administration of anti-Gr-1 monoclonal antibody in vivo

During investigating the expression of Gr-1 antigen on various subsets of mouse spleen cells, we found that Gr-1 was expressed on memory-type CD8(+)CD44(high)CD62L(high) T cells in addition to granulocytes. Intraperitoneal administration of anti-Gr-1 mAb caused almost complete elimination of Ly-6C(+) memory-type CD8(+) T cells as well as Ly-6G(+) granulocytes. Anti-Gr-1 mAb-treated mouse spleen cells exhibited greatly reduced IFN-gamma production in response to anti-CD3 mAb both in vitro and in vivo. This reduced cytokine production appeared to be derived from elimination of IFN-gamma-producing Gr-1(+)CD8(+) T cells. Indeed, CD8(+) T cells with IFN-gamma-producing activity and cytotoxicity were generated from isolated Gr-1(+)CD8(+) cells but not from Gr-1(-)CD8(+) T cells. We also demonstrated that therapeutic effect of MBL-2 tumor-immunized spleen cells was greatly reduced by anti-Gr-1 mAb-treatment. Thus, we initially demonstrated that anti-Gr-1 mAb might become a good tool to investigate a precise role for memory-type CD8(+) T cells in vivo.     

Histamine deficiency promotes inflammation-associated carcinogenesis through reduced myeloid maturation and accumulation of CD11b+Ly6G+ immature myeloid cells

Histidine decarboxylase (HDC), the unique enzyme responsible for histamine generation, is highly expressed in myeloid cells, but its function in these cells is poorly understood. Here we show that Hdc-knockout mice show a high rate of colon and skin carcinogenesis. Using Hdc-EGFP bacterial artificial chromosome (BAC) transgenic mice in which EGFP expression is controlled by the Hdc promoter, we show that Hdc is expressed primarily in CD11b(+)Ly6G(+) immature myeloid cells (IMCs) that are recruited early on in chemical carcinogenesis. Transplant of Hdc-deficient bone marrow to wild-type recipients results in increased CD11b(+)Ly6G(+) cell mobilization and reproduces the cancer susceptibility phenotype of Hdc-knockout mice. In addition, Hdc-deficient IMCs promote the growth of tumor allografts, whereas mouse CT26 colon cancer cells downregulate Hdc expression through promoter hypermethylation and inhibit myeloid cell maturation. Exogenous histamine induces the differentiation of IMCs and suppresses their ability to support the growth of tumor allografts. These data indicate key roles for Hdc and histamine in myeloid cell differentiation and CD11b(+)Ly6G(+) IMCs in early cancer development.     

Subpopulations of mouse blood monocytes differ in maturation stage and inflammatory response

Blood monocytes are well-characterized precursors for macrophages and dendritic cells. Subsets of human monocytes with differential representation in various disease states are well known. In contrast, mouse monocyte subsets have been characterized minimally. In this study we identify three subpopulations of mouse monocytes that can be distinguished by differential expression of Ly-6C, CD43, CD11c, MBR, and CD62L. The subsets share the characteristics of extensive phagocytosis, similar expression of M-CSF receptor (CD115), and development into macrophages upon M-CSF stimulation. By eliminating blood monocytes with dichloromethylene-bisphosphonate-loaded liposomes and monitoring their repopulation, we showed a developmental relationship between the subsets. Monocytes were maximally depleted 18 h after liposome application and subsequently reappeared in the circulation. These cells were exclusively of the Ly-6C(high) subset, resembling bone marrow monocytes. Serial flow cytometric analyses of newly released Ly-6C(high) monocytes showed that Ly-6C expression on these cells was down-regulated while in circulation. Under inflammatory conditions elicited either by acute infection with Listeria monocytogenes or chronic infection with Leishmania major, there was a significant increase in immature Ly-6C(high) monocytes, resembling the inflammatory left shift of granulocytes. In addition, acute peritoneal inflammation recruited preferentially Ly-6C(med-high) monocytes. Taken together, these data identify distinct subpopulations of mouse blood monocytes that differ in maturation stage and capacity to become recruited to inflammatory sites.     

Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of suppressive myeloid progenitors for NO production

Systemic infections caused by fungi after cytoreductive therapies are especially difficult to deal with in spite of currently available antimicrobials. However, little is known about the effects of fungi on the immune system of immunosuppressed hosts. We have addressed this by studying the in vitro T cell responses after systemic infection with Candida albicans in cyclophosphamide-treated mice. After cyclophosphamide treatment, a massive splenic colonization of the spleens, but not lymph nodes, by immature myeloid progenitor (Ly-6G(+)CD11b(+))cells is observed. These cells are able to suppress proliferation of T lymphocytes via a nitric oxide (NO)-dependent mechanism. Systemic infection with a sublethal dose of C. albicans did not cause immunosuppression per se but strongly increased NO-dependent suppression in cyclophosphamide-treated mice, by selective priming of suppressive myeloid progenitors (Ly-6G(+)CD11b(+)CD31(+)CD40(+)WGA(+)CD117(low/-)CD34(low/-)) for iNOS protein expression. The results indicate that systemic C. albicans infection can augment the effects of immunosuppressive therapies by promoting functional changes in immunosuppressive cells.     

Identification and characterization of novel bone marrow myeloid DEC205+Gr-1+ cell subsets that differentially express chemokine and TLRs

Bone marrow-derived immunomodulatory cytokines impart a critical function in the regulation of innate immune responses and hemopoiesis. However, the source of immunomodulatory cytokines in murine bone marrow and the cellular immune mechanisms that control local cytokine secretion remain poorly defined. Herein, we identified a population of resident murine bone marrow myeloid DEC205(+)CD11c(-)B220(-)Gr1(+)CD8alpha(-)CD11b(+) cells that respond to TLR2, TLR4, TLR7, TLR8, and TLR9 agonists as measured by the secretion of proinflammatory and anti-inflammatory cytokines in vitro. Phenotypic and functional analyses revealed that DEC205(+)CD11b(+)Gr-1(+) bone marrow cells consist of heterogeneous populations of myeloid cells that can be divided into two main cell subsets based on chemokine and TLR gene expression profile. The DEC205(+)CD11b(+)Gr-1(low) cell subset expresses high levels of TLR7 and TLR9 and was the predominant source of IL-6, TNF-alpha, and IL-12 p70 production following stimulation with the TLR7 and TLR9 agonists CpG and R848, respectively. In contrast, the DEC205(+)CD11b(+)Gr-1(high) cell subset did not respond to CpG and R848 stimulation, which correlated with their lack of TLR7 and TLR9 expression. Similarly, a differential chemokine receptor expression profile was observed with higher expression of CCR1 and CXCR2 found in the DEC205(+)CD11(+)Gr-1(high) cell subset. Thus, we identified a previously uncharacterized population of resident bone marrow cells that may be implicated in the regulation of local immune responses in the bone marrow.     

Ly-6A.2 expression regulates antigen-specific CD4+ T cell proliferation and cytokine production.

Ly-6 proteins appear to serve cell adhesion and cell signaling function, but the precise role of Ly-6A.2 in CD4+ T lymphocytes is still unclear. Overexpression of Ly-6A.2 in T lymphocytes has allowed us to analyze the influence of elevated Ly-6A.2 expression on T cell function. In this study we report reduced proliferation of CD4+ T cells overexpressing Ly-6A.2 in response to a peptide Ag. Moreover, the Ly-6A.2-overexpressing CD4+ cells generated elevated levels of IL-4, a key factor that propels the differentiation of naive CD4+ T cells into Th2 subset. The hyporesponsiveness of Ly-6A.2 transgenic CD4+ T cells is dependent on the interaction of Ly-6A.2 T cells with the APCs and can be reversed by blocking the interaction between Ly-6A.2 and a recently reported candidate ligand. Overexpression of Ly-6A.2 in CD4+ T cells reduced their Ca(2+) responses to TCR stimulation, therefore suggesting effects of Ly-6A.2 signaling on membrane proximal activation events. In contrast to the observed Ag-specific hyporesponsiveness, the Ly-6A.2 transgenic CD4+ T cells produced IL-4 independent of the interactions between Ly-6A.2 and the candidate Ly-6A.2 ligand. Our results suggest that 1) interaction of Ly-6A.2 with a candidate ligand regulates clonal expansion of CD4+ Th cells in response to an Ag (these results also provide further functional evidence for presence of Ly-6A.2 ligand on APC); and 2) Ly-6A.2 expression on CD4+ T cells promotes production of IL-4, a Th2 differentiation factor.