CCR2-deficient Mice Research Articles

The Critical Role of Chemokine (C-C Motif) Receptor 2-Positive Monocytes in Autoimmune Cholangitis.

The therapy of primary biliary cholangitis (PBC) has lagged behind other autoimmune diseases despite significant improvements in our understanding of both immunological and molecular events that lead to loss of tolerance to the E2 component of pyruvate dehydrogenase, the immunodominant autoepitope of PBC. It is well known that Ly6Chi monocytes are innate immune cells infiltrating inflammatory sites that are dependent on the expression of C–C motif chemokine receptor 2 (CCR2) for emigration from bone marrow. Importantly, humans with PBC have a circulating monocyte pro-inflammatory phenotype with macrophage accumulation in portal tracts. We have taken advantage of an inducible chemical xenobiotic model of PBC and recapitulated the massive infiltration of monocytes to portal areas. To determine the clinical significance, we immunized both CCR2-deficient mice and controls with 2OA-BSA and noted that CCR2 deficiency is protective for the development of autoimmune cholangitis. Importantly, because of the therapeutic potential, we focused on inhibiting monocyte infiltration through the use of cenicriviroc (CVC), a dual chemokine receptor CCR2/CCR5 antagonist shown to be safe in human trials. Importantly, treatment with CVC resulted in amelioration of all aspects of disease severity including serum total bile acids, histological severity score, and fibrosis stage. In conclusion, our results indicate a major role for Ly6Chi monocytes and for CCR2 in PBC pathogenesis and suggest that inhibition of this axis by CVC should be explored in humans through the use of clinical trials.

The chemokine receptor CCR7 is a promising target for rheumatoid arthritis therapy.

The chemokine receptor CCR7 and its ligands CCL19 and CCL21 guide the homing and positioning of dendritic and T cells in lymphoid organs, thereby contributing to several aspects of adaptive immunity and immune tolerance. In the present study, we investigated the role of CCR7 in the pathogenesis of collagen-induced arthritis (CIA). By using a novel anti-human CCR7 antibody and humanized CCR7 mice, we evaluated CCR7 as a target in this autoimmune model of rheumatoid arthritis (RA). Ccr7-deficient mice were completely resistant to CIA and presented severely impaired antibody responses to collagen II (CII). Selective CCR7 expression on dendritic cells restored arthritis severity and anti-CII antibody titers. Prophylactic and therapeutic treatment of humanized CCR7 mice with anti-human CCR7 mAb 8H3-16A12 led to complete resistance to CIA and halted CIA progression, respectively. Our data demonstrate that CCR7 signaling is essential for the induction of CIA and identify CCR7 as a potential therapeutic target in RA.

Murine macrophage chemokine receptor CCR2 plays a crucial role in macrophage recruitment and regulated inflammation in wound healing.

Macrophages play a critical role in the establishment of a regulated inflammatory response following tissue injury. Following injury, CCR2+ monocytes are recruited from peripheral blood to wound tissue, and direct the initiation and resolution of inflammation that is essential for tissue repair. In pathologic states where chronic inflammation prevents healing, macrophages fail to transition to a reparative phenotype. Using a murine model of cutaneous wound healing, we found that CCR2-deficient mice (CCR2-/- ) demonstrate significantly impaired wound healing at all time points postinjury. Flow cytometry analysis of wounds from CCR2-/- and WT mice revealed a significant decrease in inflammatory, Ly6CHi recruited monocyte/macrophages in CCR2-/- wounds. We further show that wound macrophage inflammatory cytokine production is decreased in CCR2-/- wounds. Adoptive transfer of mT/mG monocyte/macrophages into CCR2+/+ and CCR2-/- mice demonstrated that labeled cells on days 2 and 4 traveled to wounds in both CCR2+/+ and CCR2-/- mice. Further, adoptive transfer of monocyte/macrophages from WT mice restored normal healing, likely through a restored inflammatory response in the CCR2-deficient mice. Taken together, these data suggest that CCR2 plays a critical role in the recruitment and inflammatory response following injury, and that wound repair may be therapeutically manipulated through modulation of CCR2.

Infiltrating CCR2+ Monocytes Drive Lethal Immunopathology during Cryptococcal Meningoencephalitis

Abstract Cryptococcal meningoencephalitis (CM) is a major cause of CNS-related mortality worldwide. While immunosuppression can lead to cryptococcal infection, immune responses are recently also reported to promote CNS injury during CM. The role of monocytes in both anti-cryptococcal protection and the CNS pathology during CM remains unknown, but CCL2 level in CNS are linked to mortality risk in HIV-associated CM. To study the role of CCR2-axis recruited monocytes and monocyte derived cells (MDC) we evaluated outcomes of CM in WT versus CCR2-deficient mice. Fungal burdens, behavioral scores, pathology, immune parameters and mouse survival were compared. WT mice developed symptomatic disease and neurological deterioration from 3 weeks with 100% mortality by wk 5, despite a reduction in fungal burdens from wk 3 to wk 5. CCL2 production and MDC accumulation peaked at week 3, corresponding to onsets of symptoms and mouse mortality. Infiltrating MDC showed classical activation phenotypes (high level of iNOS/CD80 and low level of Arg1/CD206). In contrast, CCR2-deficient mice displayed a marked reduction in MDC recruitment relative to WT mice after infection. T cell inflammatory responses were diminished and switched from a Th1 response to mixed Th1/Th2/Th17 responses in CCR2 deficient mice. Furthermore, MDC activation shifted from M1 towards M2-type. These results show that CCR2-axis recruited MDC contributed to exuberant and detrimental Th1 inflammation. Survival of CCR2-deficient mice significantly improved despite impaired fugal clearance relative to WT mice. Taken together, we conclude that CCR2+ monocyte/MDC, while important for fungal clearance in CNS, are also critical mediators of inflammatory damage in CM.

Myeloid Cells in the Immune Response to Giardia

Abstract Infection with the protozoan parasite, Giardia, produces symptoms associated with intestinal distress, but does not induce intestinal inflammation. Long-term sequellae of this infection include malnutrition, Irritable Bowel Syndrome and Chronic Fatigue Syndrome. Previous work indicates that T-cells are critical for clearance of this parasite; however, it is unclear which cells are responsible for the activation of T-cells. As such, our goal was to examine the contribution of myeloid cells to Giardia immunity as possible T-cell activators, effector cells and/or immune regulators. Small intestinal lamina propria cells were isolated from Giardia-infected wild-type or CCR2 deficient mice. Flow cytometry was used to determine cell phenotypes and to monitor cytokine response. EdU labeling was used to examine in situ proliferation of resident macrophages. Lastly, wild-type mice were treated with an anti-CSF1R monoclonal antibody to deplete macrophage populations. Flow cytometry demonstrated that CD11b+, F4/80+, CD11cint, MHCII+, Ly6Cint, CX3CR1int cells accumulate in the small intestine and express ARG1 and NOS2 during infection. Intracellular cytokine staining revealed an increase in IL-10 with a concurrent decrease in TNF-α. Macrophage accumulation was the same in wild-type and CCR2-deficient mice, and EdU labeling suggested in situ proliferation of resident macrophages rather than recruitment of inflammatory monocytes. Mice treated with anti-CSF1R eliminated the parasites as well as control mice. This study demonstrates that macrophages with a regulatory phenotype accumulate in the small intestine during Giardia infections and may explain why intestinal inflammation is not present during Giardia infections.

Genetic Polymorphism at CCL5 Is Associated With Protection in Chagas' Heart Disease: Antagonistic Participation of CCR1+ and CCR5+ Cells in Chronic Chagasic Cardiomyopathy.

Chronic cardiomyopathy is the main clinical manifestation of Chagas disease (CD), a disease caused by Trypanosoma cruzi infection. A hallmark of chronic chagasic cardiomyopathy (CCC) is a fibrogenic inflammation mainly composed of CD8+ and CD4+ T cells and macrophages. CC-chemokine ligands and receptors have been proposed to drive cell migration toward the heart tissue of CD patients. Single nucleotide polymorphisms (SNPs) in CC-chemokine ligand and receptor genes may determine protein expression. Herein, we evaluated the association of SNPs in the CC-chemokines CCL2 (rs1024611) and CCL5 (rs2107538, rs2280788) and the CCL5/RANTES receptors CCR1 (rs3181077, rs1491961, rs3136672) and CCR5 (rs1799987) with risk and progression toward CCC. We performed a cross-sectional association study of 406 seropositive patients from endemic areas for CD in the State of Pernambuco, Northeast Brazil. The patients were classified as non-cardiopathic (A, n = 110) or cardiopathic (mild, B1, n = 163; severe, C, n = 133). Serum levels of CCL5 and CCL2/MCP-1 were elevated in CD patients but were neither associated with risk/severity of CCC nor with SNP genotypes. After logistic regression analysis with adjustment for the covariates gender and ethnicity, CCL5 −403 (rs2107538) CT heterozygotes (OR = 0.5, P-value = 0.04) and T carriers (OR = 0.5, P-value = 0.01) were associated with protection against CCC. To gain insight into the participation of the CCL5–CCR5/CCR1 axis in CCC, mice were infected with the Colombian T. cruzi strain. Increased CCL5 concentrations were detected in cardiac tissue. In spleen, frequencies of CCR1+ CD8+ T cells and CD14+ macrophages were decreased, while frequencies of CCR5+ cells were increased. Importantly, CCR1+CD14+ macrophages were mainly IL-10+, while CCR5+ cells were mostly TNF+. CCR5-deficient infected mice presented reduced TNF concentrations and injury in heart tissue. Selective blockade of CCR1 (Met-RANTES therapy) in infected Ccr5−/− mice supported a protective role for CCR1 in CCC. Furthermore, parasite antigen stimulation of CD patient blood cells increased the frequency of CCR1+CD8+ T cells and CCL5 production. Collectively, our data support that a genetic variant of CCL5 and CCR1+ cells confer protection against Chagas heart disease, identifying the CCL5-CCR1 axis as a target for immunostimulation.

Local Delivery Is Critical for Monocyte Chemotactic Protein-1 Mediated Site-Specific Murine Aneurysm Healing.

Local delivery of monocyte chemotactic protein-1 (MCP-1/CCL2) via our drug-eluting coil has been shown to promote intrasaccular aneurysm healing via an inflammatory pathway. In this study, we validate the importance of local MCP-1 in murine aneurysm healing. Whether systemic, rather than local, delivery of MCP-1 can direct site-specific aneurysm healing has significant translational implications. If systemic MCP-1 is effective, then MCP-1 could be administered as a pill rather than by endovascular procedure. Furthermore, we confirm that MCP-1 is the primary effector in our MCP-1 eluting coil-mediated murine aneurysm healing model. We compare aneurysm healing with repeated intraperitoneal MCP-1 versus vehicle injection, in animals with control poly(lactic-co-glycolic) acid (PLGA)-coated coils. We demonstrate elimination of the MCP-1-associated tissue-healing response by knockout of MCP-1 or CCR2 (MCP-1 receptor) and by selectively inhibiting MCP-1 or CCR2. Using immunofluorescent probing, we explore the cell populations found in healed aneurysm tissue following each intervention. Systemically administered MCP-1 with PLGA coil control does not produce comparable aneurysm healing, as seen with MCP-1 eluting coils. MCP-1-directed aneurysm healing is eliminated by selective inhibition of MCP-1 or CCR2 and in MCP-1-deficient or CCR2-deficient mice. No difference was detected in M2 macrophage and myofibroblast/smooth muscle cell staining with systemic MCP-1 versus vehicle in aneurysm wall, but a significant increase in these cell types was observed with MCP-1 eluting coil implant and attenuated by MCP-1/CCR2 blockade or deficiency. We show that systemic MCP-1 concurrent with PLGA-coated platinum coil implant is not sufficient to produce site-specific aneurysm healing. MCP-1 is a critical, not merely complementary, actor in the aneurysm healing pathway.

New interplay between interstitial and alveolar macrophages explains pulmonary alveolar proteinosis (PAP) induced by indium tin oxide particles

Occupational exposure to indium tin oxide (ITO) particles has been associated with the development of severe lung diseases, including pulmonary alveolar proteinosis (PAP). The mechanisms of this lung toxicity remain unknown. Here, we reveal the respective roles of resident alveolar (Siglec-Fhigh AM) and recruited interstitial (Siglec-Flow IM) macrophages contributing in concert to the development of PAP. In mice treated with ITO particles, PAP is specifically associated with IL-1α (not GM-CSF) deficiency and Siglec-Fhigh AM (not Siglec-Flow IM) depletion. Mechanistically, ITO particles are preferentially phagocytosed and dissolved to soluble In3+ by Siglec-Flow IM. In contrast, Siglec-Fhigh AM weakly phagocytose or dissolve ITO particles, but are sensitive to released In3+ through the expression of the transferrin receptor-1 (TfR1). Blocking pulmonary Siglec-Flow IM recruitment in CCR2-deficient mice reduces ITO particle dissolution, In3+ release, Siglec-Fhigh AM depletion, and PAP formation. Restoration of IL-1-related Siglec-Fhigh AM also prevented ITO-induced PAP. We identified a new mechanism of secondary PAP development according to which metal ions released from inhaled particles by phagocytic IM disturb IL-1α-dependent AM self-maintenance and, in turn, alveolar clearance.

CCR5-Dependent Homing of T Regulatory Cells to the Tumor Microenvironment Contributes to Skin Squamous Cell Carcinoma Development.

Squamous cell carcinoma (SCC) is one of the most common human cancers worldwide. Recent studies show that regulatory T cells (Treg) have a critical role in the modulation of an antitumor immune response, and consequently the SCC development. Because the accumulation of Tregs at the tumor site is, in part, due to selective recruitment through CCR5- and CCR5-associated chemokines, we investigated the role of CCR5 in the SCC development. Our findings showed that CCR5-deficient mice (CCR5KO) were efficient in controlling papilloma's incidence when compared with wild-type mice. Analysis of tumor lesions in wild-type (WT) and CCR5KO mice revealed that lack of CCR5 lead to significant reduction in frequency of Tregs and increased of CD4 T cells into the tumors. Moreover, the adoptive transfer of naturally occurring Tregs CD4+CD25+CCR5+, CD4+CD25-CCR5+ or CD8+CCR5+ conventional T cells to CCR5KO mice resulted in an increased papilloma incidence. Interestingly, adoptive transfer of WT CD4+CD25+CCR5+ cells to CCR5KO mice induced more undifferentiated SCC lesions, characterized by higher infiltration of macrophages and dendritic cells. In this study, we also demonstrated that Treg migration to the tumor microenvironment is mediated by CCR5, and these cells are promoting tumor growth via inhibition of antitumor cells such as cytotoxic CD8+ T cells. Our findings reinforce the therapeutic potential of CCR5 inhibition for cancer treatment, and indicate an attractive approach for SCC treatment. Mol Cancer Ther; 16(12); 2871-80. ©2017 AACR.

The HIV co-receptor CCR5 regulates osteoclast function

C–C chemokine receptor 5 (CCR5) is a co-receptor of HIV. Epidemiological findings suggest that the functional loss of CCR5 is correlated with a lower incidence of bone-destructive diseases as well as of HIV transmission. However, it is not clear whether CCR5 is involved in regulation of the function of bone cells, in addition to that of immune cells. Here we show that blockade of CCR5 using specific antibodies impairs human osteoclast function in vitro. Ccr5-deficient (Ccr5−/−) mice presented with dysfunctional osteoclasts and were resistant to osteoporosis induced by receptor activator of nuclear factor kappa-B ligand (RANKL), which triggers osteoporosis independently of inflammatory and immunomodulatory pathways. Furthermore, Ccr5 deficiency impairs the cellular locomotion and bone-resorption activity of osteoclasts, which is associated with the disarrangement of podosomes and adhesion complex molecules including Pyk2. Overall, the data provides evidence that CCR5 has an essential role in bone-destructive conditions through the functional regulation of osteoclasts.

NALT M cells are important for immune induction for the common mucosal immune system.

Nasopharynx-associated lymphoid tissue (NALT) is one of the major constituents of the mucosa-associated lymphoid tissue (MALT), and has the ability to induce antigen-specific immune responses. However, the molecular mechanisms responsible for antigen uptake from the nasal cavity into the NALT remain largely unknown. Immunohistochemical analysis showed that CCL9 and CCL20 were co-localized with glycoprotein 2 (GP2) in the epithelium covering NALT, suggesting the existence of M cells in NALT. In analogy with the reduced number of Peyer's patch M cells in CCR6-deficient mice, the number of NALT M cells was drastically decreased in CCR6-deficient mice compared with the wild-type mice. Translocation of nasally administered Salmonella enterica serovar Typhimurium into NALT via NALT M cells was impaired in CCR6-deficient mice, whereas S. Typhimurium demonstrated consistent co-localization with NALT M cells in wild-type mice. When wild-type mice were nasally administered with an attenuated vaccine strain of S. Typhimurium, the mice were protected from a subsequent challenge with wild-type S. Typhimurium. Antigen-specific fecal and nasal IgA was detected after nasal immunization with the attenuated vaccine strain of S. Typhimurium only in wild-type mice but not in CCR6-deficient mice. Taken together, these observations demonstrate that NALT M cells are important as a first line of defense against infection by enabling activation of the common mucosal immune system (CMIS).

Pericardial Adipose Tissue Regulates Granulopoiesis, Fibrosis, and Cardiac Function After Myocardial Infarction.

The pericardial adipose tissue (AT) contains a high density of lymphoid clusters. It is unknown whether these clusters play a role in post-myocardial infarction (MI) inflammatory responses and cardiac outcome. Lymphoid clusters were examined in epicardial AT of humans with or without coronary artery disease. Murine pericardial lymphoid clusters were visualized in mice subjected to coronary artery ligation. To study the relevance of pericardial clusters during inflammatory responses after MI, we surgically removed the pericardial AT and performed B-cell depletion and granulocyte-macrophage colony-stimulating factor blockade. Leukocytes in murine hearts, pericardial AT, spleen, mediastinal lymph nodes, and bone marrow were quantified by flow cytometry. Cannabinoid receptor CB2 (CB2-/-) mice were used as a model for enhanced B-cell responses. The effect of impaired dendritic cell (DC) trafficking on pericardial AT inflammatory responses was tested in CCR7-/- mice subjected to MI. Cardiac fibrosis and ventricular function were assessed by histology and echocardiography. We identified larger B-cell clusters in epicardial AT of human patients with coronary artery disease in comparison with controls without coronary artery disease. Infarcted mice also had larger pericardial clusters and 3-fold upregulated numbers of granulocyte-macrophage colony-stimulating factor-producing B cells within pericardial AT, but not spleen or lymph nodes. This was associated with higher DC and T-cell counts in pericardial AT, which outnumbered DCs and T cells in lymph nodes. Analysis of DC maturation markers, tracking experiments with fluorescently labeled cells, and use of CCR7-deficient mice suggested that activated DCs migrate from infarcts into pericardial AT via CCR7. B-cell depletion or granulocyte-macrophage colony-stimulating factor neutralization inhibited DC and T-cell expansion within pericardial AT, and translated into reduced bone marrow granulopoiesis and cardiac neutrophil infiltration 3 days after MI. The relevance of the pericardial AT in mediating all these effects was confirmed by removal of pericardial AT and ex vivo coculture with pericardial AT and granulocyte progenitors. Finally, enhanced fibrosis and worsened ejection fraction in CB2-/- mice were limited by pericardial AT removal. Our findings unveil a new mechanism by which the pericardial AT coordinates immune cell activation, granulopoiesis, and outcome after MI.

CCR7 Is Important for Mesangial Cell Physiology and Repair.

The homeostatic chemokine receptor CCR7 serves as key molecule in lymphocyte homing into secondary lymphoid tissues. Previous experiments from our group identified CCR7 also to be expressed by human mesangial cells. Exposing cultured human mesangial cells to the receptor ligand CCL21 revealed a positive effect on these cells regarding proliferation, migration, and survival. In the present study, we localized CCR7 and CCL21 during murine nephrogenesis. Analyzing wild-type and CCR7 deficient (CCR7-/-) mice, we observed a retarded glomerulogenesis during renal development and a significantly decreased mesangial cellularity in adult CCR7-/- mice, as a consequence of less mesangial cell proliferation between embryonic day E17.5 and week 5 postpartum. Cell proliferation assays and cell-wounding experiments confirmed reduced proliferative and migratory properties of mesangial cells cultured from CCR7-/- kidneys. To further emphasize the role of CCR7 as important factor for mesangial biology, we examined the chemokine receptor expression in rats after induction of a mesangioproliferative glomerulonephritis. Here, we demonstrated for the first time that extra- and intraglomerular mesangial cells that were CCR7-negative in control rats exhibited a strong CCR7 expression during the phase of mesangial repopulation and proliferation.

CCR7 Deficiency Allows Accelerated Clearance of Chlamydia from the Female Reproductive Tract.

Immune mechanisms responsible for pathogen clearance from the female reproductive tract (FRT) are incompletely defined; in particular, the contribution of lymphocyte trafficking to this process is unclear. CCR7-deficient mice have profoundly altered lymphocyte recirculation and display ectopic formation of lymphocyte aggregates within mucosal nonlymphoid tissues, including the FRT. In this study, we investigated how altered lymphocyte distribution in CCR7-deficient mice would affect host responses to Chlamydia muridarum within the reproductive tract. As expected, CCR7-deficient mice exhibited reduced lymphocyte trafficking to lymph nodes and a corresponding increase in T cell populations within the FRT. After intravaginal infection with Chlamydia, CCR7-deficient mice displayed markedly reduced Ag-specific CD4 T cell responses within the local draining iliac lymph nodes, yet robust Th1 and Th17 responses were prominent in the FRT. In addition, Chlamydia-specific Ab responses were dysregulated in CCR7-deficient mice, displaying an unexpected increase in the systemic IgA responses. Importantly, prominent mucosal immune responses in CCR7-deficient mice increased the efficiency of bacteria clearance from the FRT while reducing tissue-associated inflammation and pathology. Thus, increased numbers of lymphocytes within the FRT result in pathogen clearance with reduced immune-mediated pathology.

CCR6 promotes steady-state mononuclear phagocyte associationwith the intestinal epithelium, imprinting and immune surveillance.

The intestinal lamina propria (LP) contains antigen-presenting cells with features of dendritic cells and macrophages, collectively referred to as mononuclear phagocytes (MNPs). Association of MNPs with the epithelium is thought to play an important role in multiple facets of intestinal immunity including imprinting MNPs with the ability to induce IgA production, inducing the expression of gut homing molecules on T cells, facilitating the capture of luminal antigens and microbes, and subsequent immune responses in the mesenteric lymph node (MLN). However, the factors promoting this process in the steady state are largely unknown, and invivo models to test and confirm the importance of LP-MNP association with the epithelium for these outcomes are unexplored. Evaluation of epithelial expression of chemoattractants in mice where MNP-epithelial associations were impaired suggested CCL20 as a candidate promoting epithelial association. Expression of CCR6, the only known receptor for CCL20, was required for MNPs to associate with the epithelium. LP-MNPs from CCR6-/- mice did not display defects in acquiring antigen and stimulating T-cell responses in exvivo assays or in responses to antigen administered systemically. However, LP-MNPs from CCR6-deficient mice were impaired at acquiring luminal and epithelial antigens, inducing IgA production in B cells, inducing immune responses in the MLN, and capturing and trafficking luminal commensal bacteria to the MLN. These findings identify a crucial role for CCR6 in promoting LP-MNPs to associate with the intestinal epithelium in the steady state to perform multiple functions promoting gut immune homeostasis.

Chemokine receptor-7 (CCR7) deficiency leads to delayed development of joint damage and functional deficits in a murine model of osteoarthritis.

Elevated chemokine receptor Ccr7 is observed in knee osteoarthritis (OA) and associated with severity of symptoms. In this study, we confirmed that CCR7 protein expression is elevated in synovial tissue from OA patients by immunohistochemical staining. We then investigated whether Ccr7 deficiency impacted structural and functional joint degeneration utilizing a murine model of OA. OA-like disease was induced in male C57BL/6 and Ccr7-deficient (Ccr7-/- ) mice by destabilization of the medial meniscus (DMM). Functional deficits were measured by computer integrated monitoring of spontaneous activity every 4 weeks after DMM surgery up 16 weeks. Joint degeneration was evaluated at 6 and 19 weeks post-surgery by histopathology, and subchondral bone changes analyzed by microCT. Results showed reduction in locomotor activities in DMM-operated C57BL/6 mice by 8 weeks, while activity decreases in Ccr7-/- mice were delayed until 16 weeks. Histopathologic evaluation showed minimal protection from early cartilage degeneration (p = 0.06) and osteophytosis (p = 0.04) in Ccr7-/- mice 6 weeks post-DMM compared to C57BL/6 controls, but not at 19 weeks. However, subchondral bone mineral density (p = 0.03) and histologic sclerosis (p = 0.02) increased in response to surgery in C57BL/6 mice at 6 weeks, while Ccr7-/- mice were protected from these changes. Our results are the first to demonstrate a role for Ccr7 in early development of functional deficits and subchondral bone changes in the DMM model. Understanding the mechanism of Ccr7 receptor signaling in the initiation of joint pathology and disability will inform the development of innovative therapies to slow symptomatic OA development after injury. Published 2017. This article is a U.S. Government work and is in the public domain in the USA. J Orthop Res 36:864-875, 2018.

CCR5 Is Involved in Interruption of Pregnancy in Mice Infected with Toxoplasma gondii during Early Pregnancy.

Toxoplasmosis can cause abortion in pregnant humans and other animals; however, the mechanism of abortion remains unknown. C-C chemokine receptor type 5 (CCR5) is essential for host defense against Toxoplasma gondii infection. To investigate the relationship between CCR5 and abortion in toxoplasmosis, we inoculated wild-type and CCR5-deficient (CCR5-/-) mice with T. gondii tachyzoites intraperitoneally on day 3 of pregnancy (embryonic day 3 [E3]). The pregnancy rate decreased as pregnancy progressed in infected wild-type mice. Histopathologically, no inflammatory lesions were observed in the fetoplacental tissues. Although wild-type mice showed a higher parasite burden at the implantation sites than did CCR5-/- mice at E6 (3 days postinfection [dpi]), T. gondii antigen was detected only in the uterine tissue and not in the fetoplacental tissues. At E8 (5 dpi), the embryos in infected wild-type mice showed poor development compared with those of infected CCR5-/- mice, and apoptosis was observed in poorly developed embryos. Compared to uninfected mice, infected wild-type mice showed increased CCR5 expression at the implantation site at E6 and E8. Furthermore, analyses of mRNA expression in the uterus of nonpregnant and pregnant mice suggested that a lack of the CCR5 gene and the downregulation of tumor necrosis factor alpha (TNF-α) and CCL3 expression at E6 (3 dpi) are important factors for the maintenance of pregnancy following T. gondii infection. These results suggested that CCR5 signaling is involved in embryo loss in T. gondii infection during early pregnancy and that apoptosis is associated with embryo loss rather than direct damage to the fetoplacental tissues.

Transcutaneous immunization with a novel imiquimod nanoemulsion induces superior T cell responses and virus protection

BackgroundTranscutaneous immunization (TCI) is a novel vaccination strategy utilizing the skin associated lymphatic tissue to induce immune responses. TCI using a cytotoxic T lymphocyte (CTL) epitope and the Toll-like receptor 7 (TLR7) agonist imiquimod mounts strong CTL responses by activation and maturation of skin-derived dendritic cells (DCs) and their migration to lymph nodes. However, TCI based on the commercial formulation Aldara only induces transient CTL responses that needs further improvement for the induction of durable therapeutic immune responses. ObjectiveTherefore we aimed to develop a novel imiquimod solid nanoemulsion (IMI-Sol) for TCI with superior vaccination properties suited to induce high quality T cell responses for enhanced protection against infections. MethodsTCI was performed by applying a MHC class I or II restricted epitope along with IMI-Sol or Aldara (each containing 5% Imiquimod) on the shaved dorsum of C57BL/6, IL-1R, Myd88, Tlr7 or Ccr7 deficient mice. T cell responses as well as DC migration upon TCI were subsequently analyzed by flow cytometry. To determine in vivo efficacy of TCI induced immune responses, CTL responses and frequency of peptide specific T cells were evaluated on day 8 or 35 post vaccination and protection in a lymphocytic choriomeningitis virus (LCMV) infection model was assessed. ResultsTCI with the imiquimod formulation IMI-Sol displayed equal skin penetration of imiquimod compared to Aldara, but elicited superior CD8+ as well as CD4+ T cell responses. The induction of T-cell responses induced by IMI-Sol TCI was dependent on the TLR7/MyD88 pathway and independent of IL-1R. IMI-Sol TCI activated skin-derived DCs in skin-draining lymph nodes more efficiently compared to Aldara leading to enhanced protection in a LCMV infection model. ConclusionOur data demonstrate that IMI-Sol TCI can overcome current limitations of previous imiquimod based TCI approaches opening new perspectives for transcutaneous vaccination strategies and allowing the use of this enhanced cutaneous drug-delivery system to be tailored for the improved prevention and treatment of infectious diseases and cancers.

215 Cardiac macrophage infiltration during chronic kidney disease accelerates cardiovascular disease

The relationship between chronic kidney disease (CKD) and increasing rates of cardiovascular disease and mortality is complex, but is important due to the massive increased risk of cardiovascular events noted...

The Intersection of Immune Responses, Microbiota and Pathogenesis in Giardiasis

Abstract Infection with the protozoan parasite, Giardia, can produce a range of symptoms including diarrhea, severe cramps, and nutrient malabsorption. Our lab and others have previously indicated that immune responses can contribute to pathogenesis. It has also been reported that intestinal microbiota can affect the ability of the parasite to colonize laboratory mice. Our goal was to determine if intestinal immune cells were activated following infection and how the microbiota interacted with intestinal immune responses. Wild-type or CCR2 deficient mice were treated with a cocktail of antibiotics and infected with the GS strain. Control mice were not treated with antibiotics and/or were not infected. Changes in the intestinal microbiome and immune responses were determined using 16S sequencing and flow cytometry of isolated lamina propria and intraepithelial lymphocyte populations, respectively. Sucrase activity was also measured in mucosal lysates. Mice treated with antibiotics or infected with Giardia exhibited shifts in their intestinal microbiomes. With or without antibiotics, we observed an increase in the frequency of CD4+ T cells in the lamina propria at day 7 post-infection. We also observed an increase in macrophage populations and activated CD8+ T cells, but only in mice not treated with antibiotics. Macrophage populations increased in wild-type and CCR2-deficient mice, and EdU labeling suggested in situ proliferation of resident macrophages. Finally, sucrase deficiency correlated with activation of CD8+ T cells. Together these data indicate that the intestinal microbiota contribute to immune cell activation during infections and suggest that the microbiota may contribute to different infection outcomes.