CCR2-antagonist prophylaxis reduces pulmonary immune pathology and markedly improves survival during influenza infection

Infection with influenza virus induces severe pulmonary immune pathology that leads to substantial human mortality. Although antiviral therapy is effective in preventing infection, no current therapy can prevent or treat influenza-induced lung injury. Previously, we reported that influenza-induced pulmonary immune pathology is mediated by inflammatory monocytes trafficking to virus-infected lungs via CCR2 and that influenza-induced morbidity and mortality are reduced in CCR2-deficient mice. In this study, we evaluated the effect of pharmacologically blocking CCR2 with a small molecule inhibitor (PF-04178903) on the entry of monocytes into lungs and subsequent morbidity and mortality in influenza-infected mice. Subcutaneous injection of mice with PF-04178903 was initiated 1 d prior to infection with influenza strain H1N1A/Puerto Rico/8/34. Compared with vehicle controls, PF-04178903-treated mice demonstrated a marked reduction in mortality (75 versus 0%) and had significant reductions in weight loss and hypothermia during subsequent influenza infection. Drug-treated mice also displayed significant reductions in bronchoalveolar lavage fluid total protein, albumin, and lactose dehydrogenase activity. Administration of PF-04178903 did not alter viral titers, severity of secondary bacteria infections (Streptococcus pneumoniae), or levels of anti-influenza-neutralizing Abs. Drug-treated mice displayed an increase in influenza nucleoprotein-specific cytotoxic T cell activity. Our results suggest that CCR2 antagonists may represent an effective prophylaxis against influenza-induced pulmonary immune pathology.     

TRAIL+ monocytes and monocyte‐related cells cause lung damage and thereby increase susceptibility to influenza–Streptococcus pneumoniae coinfection

Streptococcus pneumoniae coinfection is a major cause of influenza-associated mortality; however, the mechanisms underlying pathogenesis or protection remain unclear. Using a clinically relevant mouse model, we identify immune-mediated damage early during coinfection as a new mechanism causing susceptibility. Coinfected CCR2^−/− mice lacking monocytes and monocyte-derived cells control bacterial invasion better, show reduced epithelial damage and are overall more resistant than wild-type controls. In influenza-infected wild-type lungs, monocytes and monocyte-derived cells are the major cell populations expressing the apoptosis-inducing ligand TRAIL. Accordingly, anti-TRAIL treatment reduces bacterial load and protects against coinfection if administered during viral infection, but not following bacterial exposure. Post-influenza bacterial outgrowth induces a strong proinflammatory cytokine response and massive inflammatory cell infiltrate. Depletion of neutrophils or blockade of TNF-α facilitate bacterial outgrowth, leading to increased mortality, demonstrating that these factors aid bacterial control. We conclude that inflammatory monocytes recruited early, during the viral phase of coinfection, induce TRAIL-mediated lung damage, which facilitates bacterial invasion, while TNF-α and neutrophil responses help control subsequent bacterial outgrowth. We thus identify novel determinants of protection versus pathology in influenza–Streptococcus pneumoniae coinfection.     

Monocytes are potent facilitators of alveolar neutrophil emigration during lung inflammation: role of the CCL2-CCR2 axis

Coordinated neutrophil and monocyte recruitment is a characteristic feature of acute lung inflammatory responses. We investigated the role of monocyte chemotactic protein-1 (CCL2, JE) and the chemokine receptor CCR2 in regulating alveolar leukocyte traffic. Groups of wild-type (WT) mice, CCR2-deficient mice, lethally irradiated CCR2-deficient and WT mice that were reciprocally bone marrow transplanted (chimeric CCR2 deficient and WT, respectively), chimeric CCR2-deficient mice with an enriched CCR2(+) alveolar macrophage population, and CCR2-deficient mice transfused with CCR2(+) mononuclear cells were treated with intratracheal CCL2 and/or Escherichia coli endotoxin. Our data show that alveolar monocyte recruitment is strictly dependent on CCR2. LPS-induced neutrophil migration to the lungs is CCR2 independent. However, when CCR2-bearing blood monocytes are present, alveolar neutrophil accumulation is accelerated and drastically amplified. We suggest that this hitherto unrecognized cooperativity between monocytes and neutrophils contributes to the strong, coordinated leukocyte efflux in lung inflammation.     

Differential regulatory function of resting and preactivated allergen-specific CD4+ CD25+ regulatory T cells in Th2-type airway inflammation

Although CD4(+)CD25(+) regulatory T (Treg) cells are known to suppress Th1 cell-mediated immune responses, their effect on Th2-type immune responses remains unclear. In this study we examined the role of Treg cells in Th2-type airway inflammation in mice. Depletion and reconstitution experiments demonstrated that the Treg cells of naive mice effectively suppressed the initiation and development of Th2-driven airway inflammation. Despite effective suppression of Th2-type airway inflammation in naive mice, adoptively transferred, allergen-specific Treg cells were unable to suppress airway inflammation in allergen-presensitized mice. Preactivated allergen-specific Treg cells, however, could suppress airway inflammation even in allergen-presensitized mice by accumulating in the lung, where they reduced the accumulation and proliferation of Th2 cells. Upon activation, allergen-specific Treg cells up-regulated CCR4, exhibited enhanced chemotactic responses to CCR4 ligands, and suppressed the proliferation of and cytokine production by polarized Th2 cells. Collectively, these results demonstrated that Treg cells are capable of suppressing Th2-driven airway inflammation even in allergen-presensitized mice in a manner dependent on their efficient migration into the inflammatory site and their regulation of Th2 cell activation and proliferation.     

The role of ChemR23 in the induction and resolution of cigarette smoke-induced inflammation

Chronic obstructive pulmonary disease is mainly triggered by cigarette smoke (CS) and progresses even after smoking cessation. CS induces an exaggerated influx of inflammatory cells to the bronchoalveolar space and lung parenchyma, likely resulting from a complex interplay between chemoattractants and their respective receptors. In a murine CS model of chronic obstructive pulmonary disease, we studied the importance of chemokine-like receptor ChemR23 for the induction and resolution of inflammation in CS-exposed lungs. Subacute and chronic CS exposure increased protein levels of the ChemR23 ligand and chemoattractant, chemerin, in bronchoalveolar lavage (BAL) fluid of wild-type (WT) mice. Moreover, the proinflammatory chemokines CXCL1, CCL2, and CCL20 were increased in the airways of CS-exposed WT mice, accompanied by a massive accumulation of inflammatory neutrophils and monocytes, CD11b(hi)CD103(-) and CD11b(lo)CD103(+) dendritic cells (DCs), and CD4(+) and CD8(+) T cells. The lung parenchyma of WT mice was infiltrated with inflammatory neutrophils, CD11b(hi)CD103(-) DCs, and activated CD4(+) T cells after CS exposure. CS-induced inflammation was severely attenuated in BAL fluid and lungs of ChemR23 knockout mice with regard to the induction of inflammatory chemokines and the recruitment of inflammatory cells. Neutrophils and CD8(+) T cells persisted in the airways of WT mice, as did the airway-derived conventional DCs in the mediastinal lymph nodes, for at least 14 d after smoking cessation. In the BAL fluid of CS-exposed ChemR23 knockout mice, there was a remarkable delayed accumulation of T cells 14 d after the final exposure. Our data support a role for ChemR23 in directing innate and adaptive immune cells to CS-exposed lungs.     

The pathological effects of CCR2+ inflammatory monocytes are amplified by an IFNAR1-triggered chemokine feedback loop in highly pathogenic influenza infection

Background

Highly pathogenic influenza viruses cause high levels of morbidity, including excessive infiltration of leukocytes into the lungs, high viral loads and a cytokine storm. However, the details of how these pathological features unfold in severe influenza infections remain unclear. Accumulation of Gr1 + CD11b + myeloid cells has been observed in highly pathogenic influenza infections but it is not clear how and why they accumulate in the severely inflamed lung. In this study, we selected this cell population as a target to investigate the extreme inflammatory response during severe influenza infection.

Results

We established H1N1 IAV-infected mouse models using three viruses of varying pathogenicity and noted the accumulation of a defined Gr1 + CD11b + myeloid population correlating with the pathogenicity. Herein, we reported that CCR2+ inflammatory monocytes are the major cell compartments in this population. Of note, impaired clearance of the high pathogenicity virus prolonged IFN expression, leading to CCR2+ inflammatory monocytes amplifying their own recruitment via an interferon-α/β receptor 1 (IFNAR1)-triggered chemokine loop. Blockage of IFNAR1-triggered signaling or inhibition of viral replication by Oseltamivir significantly suppresses the expression of CCR2 ligands and reduced the influx of CCR2+ inflammatory monocytes. Furthermore, trafficking of CCR2+ inflammatory monocytes from the bone marrow to the lung was evidenced by a CCR2-dependent chemotaxis. Importantly, leukocyte infiltration, cytokine storm and expression of iNOS were significantly reduced in CCR2−/− mice lacking infiltrating CCR2+ inflammatory monocytes, enhancing the survival of the infected mice.

Conclusions

Our results indicated that uncontrolled viral replication leads to excessive production of inflammatory innate immune responses by accumulating CCR2+ inflammatory monocytes, which contribute to the fatal outcomes of high pathogenicity virus infections.     

Post-BMT lung injury occurs independently of the expression of CCL2 or its receptor, CCR2, on host cells

Idiopathic pneumonia syndrome (IPS) is a significant cause of mortality post-bone marrow transplant (BMT) in humans. In our murine model, lethal pre-BMT conditioning and allogeneic T cells result in the recruitment of host antigen-presenting cells (APC) and donor T cells into the lung post-BMT concomitant with development of severe lung dysfunction. CCL2 induction is found in bronchoalveolar lavage fluid (BALF) before host monocyte influx. The major receptor for CCL2 is CCR2 present on monocytes; this interaction can play a crucial role in monocyte recruitment in inflammation. To determine whether blockade of the CCL2/CCR2 pathway could hinder host monocyte influx, lethally conditioned wild-type (WT), CCL2(-/-), or CCR2(-/-) mice were transplanted with allogeneic marrow and spleen cells. WT and (-/-) recipients exhibited equivalent lung dysfunction post-BMT. The frequencies of host macrophages as well as donor CD4(+) and CD8(+) T cells in lungs post-BMT did not differ between WT and (-/-) recipients. However, the T cell dependency of the host CD11b(+) major histocompatibility complex class II(+) cell influx was lost in CCR2(-/-) recipients. In CCR2(-/-) mice, this influx was accompanied by elevated levels of CCL20. Post-BMT BALF and sera of (-/-) mice did not reveal any decrease in cytokines or chemokines compared with WT mice. CCL2(-/-) mice had a deficiency of CCL2 in their BALF and sera post-BMT, confirming our hypothesis that CCL2 is predominantly host derived. Therefore, IPS can occur independently of host expression of CCL2 or CCR2, and compensatory mechanisms exist for regulating APC recruitment into the lung during the early post-BMT period.     

CD4+ and CD8+ T cells exhibit differential requirements for CCR7-mediated antigen transport during influenza infection

Upon encounter of viral Ags in an inflammatory environment, dendritic cells up-regulate costimulatory molecules and the chemokine receptor CCR7, with the latter being pivotal for their migration to the lymph node. By utilizing mice deficient in CCR7, we have examined the requirement of dendritic cell-mediated Ag transport from the lung to the draining lymph node for the induction of anti-influenza immune responses in vivo. We found that CCR7-mediated migration of dendritic cells was more crucial for CD8(+) T cell than CD4(+) T cell responses. While no specific CD8(+) T cell response could be detected in the airways or lymphoid tissues during the primary infection, prolonged infection in CCR7-deficient mice did result in a sustained inflammatory chemokine profile, which led to nonspecific CD8(+) T cell recruitment to the airways. The recruitment of influenza-specific CD4(+) T cells to the airways was also below levels of detection in the absence of CCR7 signaling, although a small influenza-specific CD4(+) T cell population was detectable in the draining lymph node, which was sufficient for the generation of class-switched anti-influenza Abs and a normal CD4(+) T cell memory population. Overall, our data show that CCR7-mediated active Ag transport is differentially required for CD4(+) and CD8(+) T cell expansion during influenza infection.     

The pulmonary environment promotes Th2 cell responses after nasal-pulmonary immunization with antigen alone, but Th1 responses are induced during instances of intense immune stimulation

The purpose of this study was to determine the nature of the CD4(+) Th cell responses induced after nasal-pulmonary immunization, especially those coinciding with previously described pulmonary inflammation associated with the use of the mucosal adjuvant, cholera toxin (CT). The major T cell population in the lungs of naive mice was CD4(+), and these cells were shown to be predominantly of Th2 type as in vitro polyclonal stimulation resulted in IL-4, but not IFN-gamma, production. After nasal immunization with influenza Ag alone, Th2 cytokine mRNA (IL-4 and IL-5) levels were increased, whereas there was no change in Th1 cytokine (IL-2 and IFN-gamma) mRNA expression. The use of the mucosal adjuvant, CT, markedly enhanced pulmonary Th2-type responses; however, there was also a Th1 component to the T cell response. Using in vitro Ag stimulation of pulmonary lymphocytes, influenza virus-specific cytokine production correlated with the mRNA cytokine results. Furthermore, there was a large increase in CD4(+) Th cell numbers in lungs after nasal immunization using CT, correlating with the pulmonary inflammatory infiltrate previously described. Coincidentally, both macrophage-inflammatory protein-1alpha (MIP-1alpha) and MIP-1beta mRNA expression increased in the lungs after immunization with Ag plus CT, while only MIP-1beta expression increased when mice were given influenza Ag alone. Our study suggests a mechanism to foster Th1 cell recruitment into the lung, which may impact on pulmonary immune responses. Thus, while Th2 cell responses may be prevalent in modulating mucosal immunity in the lungs, Th1 cell responses contribute to pulmonary defenses during instances of intense immune stimulation.     

The magnitude of the T cell response to a clinically significant dose of influenza virus is regulated by TRAIL

An immune response of appropriate magnitude should be robust enough to control pathogen spread but not simultaneously lead to immunopathology. Primary infection with influenza A virus (IAV) results in a localized pulmonary infection and inflammation and elicits an IAV-specific CD8 T cell immune response necessary for viral clearance. Clearance of IAV-infected cells, and recovery from infection, is mediated by perforin/granzyme B- and Fas/FasL-mediated mechanisms. We recently reported that TRAIL is another means by which IAV-specific CD8 T cells can kill IAV-infected cells. The current study examined the role of TRAIL in the pulmonary CD8 T cell response to a clinically significant IAV [A/PR/8/34 (PR8; H1N1)] infection (i.e., leads to observable, but limited, morbidity and mortality in wild-type [WT] mice). Compared with WT mice, IAV-infected Trail(-/-) mice experienced increased morbidity and mortality despite similar rates of viral clearance from the lungs. The increased morbidity and mortality in Trail(-/-) mice correlated with increased pulmonary pathology and inflammatory chemokine production. Analysis of lung-infiltrating lymphocytes revealed increased numbers of IAV-specific CD8 T cells in infected Trail(-/-) mice, which correlated with increased pulmonary cytotoxic activity and increased pulmonary expression of MIG and MIP-1α. In addition, there was decreased apoptosis and increased proliferation of IAV-specific CD8 T cells in the lungs of Trail(-/-) mice compared with WT mice. Together, these data suggest that TRAIL regulates the magnitude of the IAV-specific CD8 T cell response during a clinically significant IAV infection to decrease the chance for infection-induced immunopathology.     

Differential roles of CC chemokine ligand 2/monocyte chemotactic protein-1 and CCR2 in the development of T1 immunity

CCR2 and its major ligand, chemokine ligand 2 (CCL2)/monocyte chemotactic protein-1, have been found to influence T1/T2 immune response polarization. Our objective was to directly compare the roles of CCR2 and CCL2 in T1/T2 immune response polarization using a model of pulmonary Cryptococcus neoformans infection. Either deletion of CCR2 or treatment of wild-type mice with CCL2 neutralizing Ab produced significant and comparable reductions in macrophage and T cell recruitment into the lungs following infection. Both CCL2 neutralization and CCR2 deficiency resulted in significantly diminished IFN-gamma production, and increased IL-4 and IL-5 production by lung leukocytes (T1 to T2 switch), but only CCR2 deficiency promoted pulmonary eotaxin production and eosinophilia. In the lung-associated lymph nodes (LALN), CCL2-neutralized mice developed Ag-specific IFN-gamma-producing cells, while CCR2 knockout mice did not. LALN from CCR2 knockout mice also had fewer MHCII(+)CD11c(+) and MHCII(+)CD11b(+) cells, and produced significantly less IL-12p70 and TNF-alpha when stimulated with heat-killed yeast than LALN from wild-type or CCL2-neutralized mice, consistent with a defect in APC trafficking in CCR2 knockout mice. Neutralization of CCL2 in CCR2 knockout mice did not alter immune response development, demonstrating that the high levels of CCL2 in these mice did not play a role in T2 polarization. Therefore, CCR2 (but not CCL2) is required for afferent T1 development in the lymph nodes. In the absence of CCL2, T1 cells polarize in the LALN, but do not traffic from the lymph nodes to the lungs, resulting in a pulmonary T2 response.     

IL-10 modulates host responses and lung damage induced by Pneumocystis carinii infection

Host responses to Pneumocystis carinii infection mediate impairment of pulmonary function and contribute to the pathogenesis of pneumonia. IL-10 is known to inhibit inflammation and reduce the severity of pathology caused by a number of infectious organisms. In the present studies, IL-10-deficient (IL-10 knockout (KO)) mice were infected with P. carinii to determine whether the severity of pathogenesis and the efficiency of clearance of the organisms could be altered in the absence of IL-10. The clearance kinetics of P. carinii from IL-10 KO mice was significantly enhanced compared with that of wild-type (WT) mice. This corresponded to a more intense CD4(+) and CD8(+) T cell response as well as an earlier neutrophil response in the lungs of IL-10 KO mice. Furthermore, IL-12, IL-18, and IFN-gamma were found in the bronchoalveolar lavage fluids at earlier time points in IL-10 KO mice suggesting that alveolar macrophages were activated earlier than in WT mice. However, when CD4(+) cells were depleted from P. carinii-infected IL-10 KO mice, the ability to enhance clearance was lost. Furthermore, CD4-depleted IL-10 KO mice had significantly more lung injury than CD4-depleted WT mice even though the intensity of the inflammatory responses was similar. This was characterized by increased vascular leakage, decreased oxygenation, and decreased arterial pH. These data indicate that IL-10 down-regulates the immune response to P. carinii in WT mice; however, in the absence of CD4(+) T cells, IL-10 plays a critical role in controlling lung damage independent of modulating the inflammatory response.     

Autocrine type I IFN and contact with endothelium promote the presentation of influenza A virus by monocyte-derived APC

Purified monocytes infected with influenza A virus do not become mature dendritic cells (DCs) and they present viral peptides poorly to autologous memory T cells. In this study, we investigated whether influenza A-infected monocytes matured to DCs with a high capacity to stimulate T cells when they were infected with influenza A virus in a model tissue setting wherein they were cocultured with endothelium grown on a type I collagen matrix. Intercellular interactions with endothelium strongly promoted the Ag-presenting capacity of monocyte-derived cells infected with influenza A virus, and the heterologous coculture system also enhanced production of IFN-alpha by monocytes in the absence of plasmacytoid cells. Production of IFN-alpha in the presence of endothelium correlated with monocyte differentiation to mature DCs and their ability to stimulate proliferation and IFN-gamma production by autologous T cells. Monocyte-derived cells that developed into migratory DCs promoted proliferation of influenza A virus-specific CD4(+) and CD8(+) cells, whereas those that developed into macrophages promoted proliferation of CD8(+) T cells only. This onset of APC activity could be partially blocked with Ab to the IFN-alphabeta receptor when monocytes were infected with UV-treated virus, but neutralizing this pathway was inconsequential when monocytes were infected with live virus. Thus, type I IFN and direct contact with endothelium promote development of influenza A virus-presenting activity in monocyte-derived cells in a setting in which this differentiation does not depend on plasmacytoid cells. However, when infected with live influenza virus, the role of type I IFN in mediating differentiation and Ag-presenting capacity is expendable, apparently due to other mechanisms of virus-mediated activation.     

Emergence of CD8+ T cells expressing NK cell receptors in influenza A virus-infected mice

Both innate and adaptive immune responses play an important role in the recovery of the host from viral infections. In the present report, a subset of cells coexpressing CD8 and NKR-P1C (NK1.1) was found in the lungs of mice infected with influenza A virus. These cells were detected at low numbers in the lungs of uninfected mice, but represented up to 10% of the total CD8(+) T cell population at day 10 postinfection. Almost all of the CD8(+)NK1.1(+) cells were CD8alphabeta(+)CD3(+)TCRalphabeta(+) and a proportion of these cells also expressed the NK cell-associated Ly49 receptors. Interestingly, up to 30% of these cells were virus-specific T cells as determined by MHC class I tetramer staining and by intracellular staining of IFN-gamma after viral peptide stimulation. Moreover, these cells were distinct from conventional NKT cells as they were also found at increased numbers in influenza-infected CD1(-/-) mice. These results demonstrate that a significant proportion of CD8(+) T cells acquire NK1.1 and other NK cell-associated molecules, and suggests that these receptors may possibly regulate CD8(+) T cell effector functions during viral infection.     

CCR1 and CC chemokine ligand 5 interactions exacerbate innate immune responses during sepsis

CCR1 has previously been shown to play important roles in leukocyte trafficking, pathogen clearance, and the type 1/type 2 cytokine balance, although very little is known about its role in the host response during sepsis. In a cecal ligation and puncture model of septic peritonitis, CCR1-deficient (CCR1(-/-)) mice were significantly protected from the lethal effects of sepsis when compared with wild-type (WT) controls. The peritoneal and systemic cytokine profile in CCR1(-/-) mice was characterized by a robust, but short-lived and regulated antibacterial response. CCR1 expression was not required for leukocyte recruitment, suggesting critical differences extant in the activation of WT and CCR1(-/-) resident or recruited peritoneal cells during sepsis. Peritoneal macrophages isolated from naive CCR1(-/-) mice clearly demonstrated enhanced cytokine/chemokine generation and antibacterial responses compared with similarly treated WT macrophages. CCR1 and CCL5 interactions markedly altered the inflammatory response in vivo and in vitro. Administration of CCL5 increased sepsis-induced lethality in WT mice, whereas neutralization of CCL5 improved survival. CCL5 acted in a CCR1-dependent manner to augment production of IFN-gamma and MIP-2 to damaging levels. These data illustrate that the interaction between CCR1 and CCL5 modulates the innate immune response during sepsis, and both represent potential targets for therapeutic intervention.     

Suppression of innate immune pathology by regulatory T cells during Influenza A virus infection of immunodeficient mice

The viral infection of higher vertebrates elicits potent innate and adaptive host immunity. However, an excessive or inappropriate immune response also may lead to host pathology that often is more severe than the direct effects of viral replication. Therefore, several mechanisms exist that regulate the magnitude and class of the immune response. Here, we have examined the potential involvement of regulatory T (Treg) cells in limiting pathology induced by influenza A virus (IAV) infection. Using lymphocyte-deficient mice as hosts, we showed that Treg cell reconstitution resulted in a significant delay in weight loss and prolonged survival following infection. The adoptively transferred Treg cells did not affect the high rate of IAV replication in the lungs of lymphocyte-deficient hosts, and therefore their disease-ameliorating effect was mediated through the suppression of innate immune pathology. Mechanistically, Treg cells reduced the accumulation and altered the distribution of monocytes/macrophages in the lungs of IAV-infected hosts. This reduction in lung monocytosis was associated with a specific delay in monocyte chemotactic protein-2 (MCP-2) induction in the infected lungs. Nevertheless, Treg cells failed to prevent the eventual development of severe disease in lymphocyte-deficient hosts, which likely was caused by the ongoing IAV replication. Indeed, using T-cell-deficient mice, which mounted a T-cell-independent B cell response to IAV, we further showed that the combination of virus-neutralizing antibodies and transferred Treg cells led to the complete prevention of clinical disease following IAV infection. Taken together, these results suggested that innate immune pathology and virus-induced pathology are the two main contributors to pathogenesis during IAV infection.     

Inflammatory blood monocytes contribute to tumor development and represent a privileged target to improve host immunosurveillance

Progressing tumors in humans and mice are frequently infiltrated by a highly heterogeneous population of inflammatory myeloid cells that contribute to tumor growth. Among these cells, inflammatory Gr-1(+) monocytes display a high developmental plasticity in response to specific microenvironmental signals, leading to diverse immune functions. These observations raise the question of the immune mechanisms by which inflammatory monocytes may contribute to tumor development. In this study, we found that adoptive transfer of normal inflammatory Gr-1(+) monocytes in tumor-bearing mice promotes tumor growth. In this tumoral environment, these monocytes can differentiate into tolerogenic dendritic cells (DCs) that produce IL-10 and potently induce regulatory T cell responses in vivo. Moreover, diverting the differentiation of Gr-1(+) monocytes into tolerogenic DCs by forced expression of IL-10 soluble receptor and IL-3 in tumor cells improves host immunosurveillance by reducing the regulatory T cell frequency and by inducing immunogenic DCs in the tumor. As a consequence, tumor growth is strongly reduced. Our findings indicate that Gr-1(+) monocytes represent a valuable target for innovative immunotherapeutic strategies against cancer.     

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.     

Type I interferon signaling regulates Ly6C(hi) monocytes and neutrophils during acute viral pneumonia in mice

Type I interferon (IFN-I) plays a critical role in the homeostasis of hematopoietic stem cells and influences neutrophil influx to the site of inflammation. IFN-I receptor knockout (Ifnar1 ^−/−) mice develop significant defects in the infiltration of Ly6C^hi monocytes in the lung after influenza infection (A/PR/8/34, H1N1). Ly6C^hi monocytes of wild-type (WT) mice are the main producers of MCP-1 while the alternatively generated Ly6C^int monocytes of Ifnar1 ^−/− mice mainly produce KC for neutrophil influx. As a consequence, Ifnar1 ^−/− mice recruit more neutrophils after influenza infection than do WT mice. Treatment of IFNAR1 blocking antibody on the WT bone marrow (BM) cells in vitro failed to differentiate into Ly6C^hi monocytes. By using BM chimeric mice (WT BM into Ifnar1 ^−/− and vice versa), we confirmed that IFN-I signaling in hematopoietic cells is required for the generation of Ly6C^hi monocytes. Of note, WT BM reconstituted Ifnar1 ^−/− chimeric mice with increased numbers of Ly6C^hi monocytes survived longer than influenza-infected Ifnar1 ^−/− mice. In contrast, WT mice that received Ifnar1 ^−/− BM cells with alternative Ly6C^int monocytes and increased numbers of neutrophils exhibited higher mortality rates than WT mice given WT BM cells. Collectively, these data suggest that IFN-I contributes to resistance of influenza infection by control of monocytes and neutrophils in the lung.