Antibodies to MHC Class II Molecules Induce Autoimmunity: Critical Role for Macrophages in the Immunopathogenesis of Obliterative Airway Disease

Previous studies have shown that intrabronchial administration of antibodies (Abs) to MHC class I resulted in development of obliterative airway disease (OAD), a correlate of chronic human lung allograft rejection. Since development of Abs specific to mismatched donor HLA class II have also been associated with chronic human lung allograft rejection, we analyzed the role of Abs to MHC class II in inducing OAD. Administration of MHC class II Abs (M5/114) to C57BL/6 mice induced the classical features of OAD even though MHC class II expression is absent de novo on murine lung epithelial and endothelial cells. The induction of OAD was accompanied by enhanced cellular and humoral immune responses to self-antigens (Collagen V and K- α1Tubulin). Further, lung-infiltrating macrophages demonstrated a switch in their phenotype predominance from MΦ1 (F4/80⁺CD11c⁺) to MΦ2 (F4/80⁺CD206⁺) following administration of Abs and prior to development of OAD. Passive administration of macrophages harvested from animals with OAD but not from naïve animals induced OAD lesions. We conclude that MHC class II Abs induces a phenotype switch of lung infiltrating macrophages from MΦ1 (F4/80⁺CD11c⁺) to MΦ2 (F4/80⁺CD206⁺) resulting in the breakdown of self-tolerance along with an increase in autoimmune Th17 response leading to OAD.     

Characterization and Identification of Subpopulations of Mononuclear Preosteoclasts Induced by TNF-α in Combination with TGF-β in Rats

Osteoclasts are unique multinucleated cells formed by fusion of preosteoclasts derived from cells of the monocyte/macrophage lineage, which are induced by RANKL. However, characteristics and subpopulations of osteoclast precursor cells are poorly understood. We show here that a combination of TNF-α, TGF-β, and M-CSF efficiently generates mononuclear preosteoclasts but not multinucleated osteoclasts (MNCs) in rat bone marrow cultures depleted of stromal cells. Using a rat osteoclast-specific mAb, Kat1, we found that TNF-α and TGF-β specifically increased Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells but not Kat1⁻c-fms⁺ cells. Kat1⁻c-fms⁺ cells appeared in early stages of culture, but Kat1⁺c-fms⁺ and Kat1⁺c-fms⁻ cells increased later. Preosteoclasts induced by TNF-α, TGF-β, and M-CSF rapidly differentiated into osteoclasts in the presence of RANKL and hydroxyurea, an inhibitor of DNA synthesis, suggesting that preosteoclasts are terminally differentiated cells. We further analyzed the expression levels of genes encoding surface proteins in bone marrow macrophages (BMM), preosteoclasts, and MNCs. Preosteoclasts expressed itgam (CD11b) and chemokine receptors CCR1 and CCR2; however, in preosteoclasts the expression of chemokine receptors CCR1 and CCR2 was not up-regulated compared to their expression in BMM. However, addition of RANKL to preosteoclasts markedly increased the expression of CCR1. In contrast, expression of macrophage antigen emr-1 (F4/80) and chemokine receptor CCR5 was down-regulated in preosteoclasts. The combination of TNF-α, TGF-β, and M-CSF induced Kat1⁺CD11b⁺ cells, but these cells were also induced by TNF-α alone. In addition, MIP-1α and MCP-1, which are ligands for CCR1 and CCR2, were chemotactic for preosteoclasts, and promoted multinucleation of preosteoclasts. Finally, we found that Kat1⁺c-fms⁺ cells were present in bone tissues of rats with adjuvant arthritis. These data demonstrate that TNF-α in combination with TGF-β efficiently generates preosteoclasts in vitro. We delineated characteristics that are useful for identifying and isolating rat preosteoclasts, and found that CCR1 expression was regulated in the fusion step in osteoclastogenesis.     

Mouse CD11b+Kupffer Cells Recruited from Bone Marrow Accelerate Liver Regeneration after Partial Hepatectomy

TNF and Fas/FasL are vital components, not only in hepatocyte injury, but are also required for hepatocyte regeneration. Liver F4/80⁺Kupffer cells are classified into two subsets; resident radio-resistant CD68⁺cells with phagocytic and bactericidal activity, and recruited radio-sensitive CD11b⁺cells with cytokine-producing capacity. The aim of this study was to investigate the role of these Kupffer cells in the liver regeneration after partial hepatectomy (PHx) in mice. The proportion of Kupffer cell subsets in the remnant liver was examined in C57BL/6 mice by flow cytometry after PHx. To examine the role of CD11b⁺Kupffer cells/Mφ, mice were depleted of these cells before PHx by non-lethal 5 Gy irradiation with or without bone marrow transplantation (BMT) or the injection of a CCR2 (MCP-1 receptor) antagonist, and liver regeneration was evaluated. Although the proportion of CD68⁺Kupffer cells did not significantly change after PHx, the proportion of CD11b⁺Kupffer cells/Mφ and their FasL expression was greatly increased at three days after PHx, when the hepatocytes vigorously proliferate. Serum TNF and MCP-1 levels peaked one day after PHx. Irradiation eliminated the CD11b⁺Kupffer cells/Mφ for approximately two weeks in the liver, while CD68⁺Kupffer cells, NK cells and NKT cells remained, and hepatocyte regeneration was retarded. However, BMT partially restored CD11b⁺Kupffer cells/Mφ and recovered the liver regeneration. Furthermore, CCR2 antagonist treatment decreased the CD11b⁺Kupffer cells/Mφ and significantly inhibited liver regeneration. The CD11b⁺Kupffer cells/Mφ recruited from bone marrow by the MCP-1 produced by CD68⁺Kupffer cells play a pivotal role in liver regeneration via the TNF/FasL/Fas pathway after PHx.     

Cell density during differentiation can alter the phenotype of bone marrow-derived macrophages

Background

Bone marrow-derived macrophages (BMDMs) are widely used primary cells for studying macrophage function. However, despite numerous protocols that are currently available, lack of a notable consensus on generating BMDMs may obscure the reliability in comparing findings from different studies or laboratories.

Findings

In this study, we addressed the effect of cell density on the resulting macrophage population. With reference to previously published methods, bone marrow cells from wild type C57BL/6 mice were plated at either 4?×?10⁵ cells or 5?×?10⁶ cells per 10 cm and cultured in 20% L-cell conditioned media for 7 days, after which they were analyzed for cell surface markers, production of proinflammatory cytokines, and responsiveness to polarizing signals. Reproducibly, cells plated at lower density gave a pure population of CD11b⁺F4/80⁺ macrophages (97.28?±?0.52%) with majority being Ly-6C^-Ly-6G^- and c-Fms⁺, while those plated at higher density produced less CD11b⁺F4/80⁺ cells and a considerably higher proportion of CD11b⁺F4/80⁺CD11c⁺ (68.72?±?2.52%) and Ly-6C^-Ly-6G⁺ (71.10?±?0.90%) cells. BMDMs derived from higher plating density also secreted less proinflammatory cytokines such as IL-6, IL-12 and TNF-α and were less phagocytic, and had a different pattern of expression for M1- and M2-related genes upon LPS or IL-4 stimulation.

Conclusions

Overall, our findings indicate that altering cell density during BMDM differentiation can give rise to distinct macrophage populations that could vary the outcome of a functional study.

     

Development of peritoneal macrophage along a dendritic cell lineage in response to uptake of oligomannose-coated liposomes

In this study, we investigate the potential of peritoneal macrophages to differentiate into dendritic cell (DCs) in response to preferential uptake of oligomannose-coated liposomes (OMLs). About 30% of peritoneal cells (PECs) preferentially took up OMLs that were administered into the peritoneal cavity. The OML-ingesting cells expressed CD11b and F4/80, but lacked CD11c expression, indicating that the OML-ingesting PECs with a CD11b^highCD11c⁻ phenotype are resident peritoneal macrophages. During in vitro cultivation, CD11c⁺ cells arose among the PECs with ingested OMLs. CD11c⁺ cells also developed among enriched peritoneal CD11b^highCD11⁻ cells from OML-treated mice, and the resulting CD11c⁺ cells expressed co-stimulatory molecules and MHC class II. In addition, OML-ingesting CD11b^highCD11c⁺ cells were found in spleen after the enriched peritoneal macrophages with ingested OMLs were transplanted in the peritoneal cavity of mice. These results show that a fraction of peritoneal macrophages can differentiate into mature DCs following uptake of OMLs.     

Potential Roles of CCR5+ CCR6+ Dendritic Cells Induced by Nasal Ovalbumin plus Flt3 Ligand Expressing Adenovirus for Mucosal IgA Responses

We assessed the role of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ dendritic cells (DCs) for induction of ovalbumin (OVA)-specific antibody (Ab) responses following mucosal immunization. Mice given nasal OVA plus an adenovirus expressing Flt3 ligand (Ad-FL) showed early expansion of CCR5⁺/CCR6⁺/CD11b⁺/CD11c⁺ DCs in nasopharyngeal-associated lymphoid tissue (NALT) and cervical lymph nodes (CLNs). Subsequently, this DC subset became resident in submandibular glands (SMGs) and nasal passages (NPs) in response to high levels of CCR-ligands produced in these tissues. CD11b⁺/CD11c⁺ DCs were markedly decreased in both CCR5^−/− and CCR6^−/− mice. Chimera mice reconstituted with bone marrow cells from CD11c-diphtheria toxin receptor (CD11c-DTR) and CCR5^−/− or CD11c-DTR and CCR6^−/− mice given nasal OVA plus Ad-FL had elevated plasma IgG, but reduced IgA as well as low anti-OVA secretory IgA (SIgA )Ab responses in saliva and nasal washes. These results suggest that CCR5⁺CCR6⁺ DCs play an important role in the induction of Ag-specific SIgA Ab responses.     

Tip-DC Development during Parasitic Infection Is Regulated by IL-10 and Requires CCL2/CCR2, IFN-γ and MyD88 Signaling

The development of classically activated monocytic cells (M1) is a prerequisite for effective elimination of parasites, including African trypanosomes. However, persistent activation of M1 that produce pathogenic molecules such as TNF and NO contributes to the development of trypanosome infection-associated tissue injury including liver cell necrosis in experimental mouse models. Aiming to identify mechanisms involved in regulation of M1 activity, we have recently documented that during Trypanosoma brucei infection, CD11b⁺Ly6C⁺CD11c⁺ TNF and iNOS producing DCs (Tip-DCs) represent the major pathogenic M1 liver subpopulation. By using gene expression analyses, KO mice and cytokine neutralizing antibodies, we show here that the conversion of CD11b⁺Ly6C⁺ monocytic cells to pathogenic Tip-DCs in the liver of T. brucei infected mice consists of a three-step process including (i) a CCR2-dependent but CCR5- and Mif-independent step crucial for emigration of CD11b⁺Ly6C⁺ monocytic cells from the bone marrow but dispensable for their blood to liver migration; (ii) a differentiation step of liver CD11b⁺Ly6C⁺ monocytic cells to immature inflammatory DCs (CD11c⁺ but CD80/CD86/MHC-II^low) which is IFN-γ and MyD88 signaling independent; and (iii) a maturation step of inflammatory DCs to functional (CD80/CD86/MHC-II^high) TNF and NO producing Tip-DCs which is IFN-γ and MyD88 signaling dependent. Moreover, IL-10 could limit CCR2-mediated egression of CD11b⁺Ly6C⁺ monocytic cells from the bone marrow by limiting Ccl2 expression by liver monocytic cells, as well as their differentiation and maturation to Tip-DCs in the liver, showing that IL-10 works at multiple levels to dampen Tip-DC mediated pathogenicity during T. brucei infection. A wide spectrum of liver diseases associates with alteration of monocyte recruitment, phenotype or function, which could be modulated by IL-10. Therefore, investigating the contribution of recruited monocytes to African trypanosome induced liver injury could potentially identify new targets to treat hepatic inflammation in general, and during parasite infection in particular.     

Chemokine receptor 5 blockade modulates macrophage trafficking in renal ischaemic-reperfusion injury

Chemokine receptor 5 (CCR5) is a pivotal regulator of macrophage trafficking in the kidneys in response to an inflammatory cascade. We investigated the role of CCR5 in experimental ischaemic-reperfusion injury (IRI) pathogenesis. To establish IRI, we clamped the bilateral renal artery pedicle for 30 min and then reperfused the kidney. We performed adoptive transfer of lipopolysaccharide (LPS)-treated RAW 264.7 macrophages following macrophage depletion in mice. B6.CCR5^−/− mice showed less severe IRI based on tubular epithelial cell apoptosis than did wild-type mice. CXCR3 expression in CD11b⁺ cells and inducible nitric oxide synthase levels were more attenuated in B6.CCR5^−/− mice. B6.CCR5^−/− mice showed increased arginase-1 and CD206 expression. Macrophage-depleted wild-type mice showed more injury than B6.CCR5^−/− mice after M1 macrophage transfer. Adoptive transfer of LPS-treated RAW 264.7 macrophages reversed the protection against IRI in wild-type, but not B6.CCR5^−/− mice. Upon knocking out CCR5 in macrophages, migration of bone marrow-derived macrophages from wild-type mice towards primary tubular epithelial cells with recombinant CCR5 increased. Phospho-CCR5 expression in renal tissues of patients with acute tubular necrosis was increased, showing a positive correlation with tubular inflammation. In conclusion, CCR5 deficiency favours M2 macrophage activation, and blocking CCR5 might aid in treating acute kidney injury.     

Characteristics and clinical significance of CD163+/CD206+M2 mono-macrophage in the bladder cancer microenvironment

The tumor microenvironment may recruit monocytes, with a protumoral macrophage phenotype (M2) that plays an important role in solid tumor progression and metastasis. Therefore, it is necessary to understand the characteristics of these cells for cancer prevention and treatment. Bladder cancer tissue samples and paracarcinoma tissues samples were collected, and the expression of CD163⁺ cells in tumor tissues was observed. Then, we observed the expression of infiltrating CD45⁺CD14⁺CD163⁺ cell subset and analyzed the molecular expressions related to immunity and angiogenesis. C57/BL6 mice were inoculated subcutaneously, and dynamic changes of CD11b⁺F4/80⁺CD206⁺ mononuclear macrophages expression for tumor-bearing mice were detected. The results showed that the proportion of CD45⁺CD14⁺CD163⁺ mono-macrophage subset infiltrated by tumor tissue was significantly higher than that in paracarcinoma tissues. In bladder cancer tissue, the expression rate of CD40 in CD45⁺CD14⁺CD163^- mono-macrophage subset was significantly lower than that in CD45⁺CD14⁺CD163⁺ mono-macrophage subset. Similar results were found in the paracarcinoma tissues. We found that, as the proportion of CD11b⁺F4/80⁺CD206⁺ mono-macrophages increased gradually, the difference was statistically significant. CD163⁺/CD206⁺ mono-macrophages in bladder cancer microenvironment are abnormally elevated, and these cells are closely related to tumor progression. CD40 may be an important molecule that exerts biological function in this subset.     

Coreceptor function of CD4 in response to the MHC class I molecule

The specificity of the T-cell receptor (TCR) and its interaction with coreceptors play a crucial role in T-cell passing through developmental checkpoints and, eventually, determine the efficiency of adaptive immunity. The genes for the α and β chains of TCR were cloned from T-cell hybridoma 1D1, which was obtained by fusion of BWZ.36CD8α cells with CD8⁺ memory cells specific for the H-2K^b MHC class I molecule. Retroviral transduction of the 1D1 TCR genes and the CD4 and CD8 coreceptor genes was used to obtain 4G4 thymoma variants that exposed the CD3/TCR complex together with CD4, CD8, or both of the coreceptors on their surface. Although the main function of CD4 is to stabilize the interaction of TCR with MHC class II molecules, CD4 was found to mediate the activation of transfected cells via TCR specific for the H-2K^b MHC class I molecule. Moreover, CD4 proved to dominate over CD8, since the response of CD4⁺CD8⁺ transfectants was suppressed by antibodies against CD4 and the A^b MHC class II molecule but not to CD8. The response of CD4⁺ transfectants was not due to a cross-reaction of 1D1 TCR with MHC class II molecules, because the transfectants did not respond to splenocytes of H-2^b knockout mice, which were defective in the assembly of the MHC class I molecule/β2 microglobulin/peptide complex and did not expose the complex on cell surface. The domination was not due to sequestration of p56^lck kinase, since CD4 devoid of the kinase-binding site was functional in 4G4 thymoma cells. The results were used to explain some features of intrathymic cell selection and assumed to provide an experimental basis for developing new methods of anticancer gene therapy.     

PPAR γ is highly expressed in F4/80 adipose tissue macrophages and dampens adipose-tissue inflammation

Macrophage infiltration into adipose tissue is a hallmark of obesity. We recently reported two phenotypically distinct subsets of adipose tissue macrophages (ATM) based on the surface expression of the glycoprotein F4/80 and responsiveness to treatment with a peroxisome proliferator-activated receptor (PPAR) γ agonist. Hence, we hypothesized that F4/80^hi and F4/80^lo ATM differentially express PPAR γ. This study phenotypically and functionally characterizes F4/80^hi and F4/80^lo ATM subsets during obesity. Changes in gene expression were also examined on sorted F4/80^lo and F4/80^hi ATM by quantitative real-time RT-PCR. We show that while F4/80^lo macrophages predominate in adipose tissue of lean mice, obesity causes accumulation of both F4/80^lo and F4/80^hi ATM. Moreover, accumulation of F4/80^hi ATM in adipose tissue is associated with impaired glucose tolerance. Phenotypically, F4/80^hi ATM express greater amounts of CD11c, MHC II, CD49b, and CX3CR1 and produce more TNF-α, MCP-1, and IL-10 than F4/80^lo ATM. Gene expression analyses of the sorted populations revealed that only the F4/80^lo population produced IL-4, whereas the F4/80^hi ATM expressed greater amounts of PPAR γ, δ, CD36 and toll-like receptor-4. In addition, the deficiency of PPAR γ in immune cells favors expression of M1 and impairs M2 macrophage marker expression in adipose tissue. Thus, PPAR γ is differentially expressed in F4/80^hi versus F4/80^low ATM subsets and its deficiency favors a predominance of M1 markers in WAT.     

High Physiological Concentrations of Progesterone Reverse Estradiol-Mediated Changes in Differentiation and Functions of Bone Marrow Derived Dendritic Cells

Female sex steroids, estradiol (E2) and progesterone (P4), play a key role in regulating immune responses in women, including dendritic cell (DC) development, and functions. Although the two hormones co-occur in the body of women throughout the reproductive years, no studies have explored their complex combinatorial effects on DCs, given their ability to regulate each other’s actions. We examined murine bone marrow derived dendritic cells (BMDC) differentiation and functions, in the presence of a wide range of physiological concentrations of each hormone, as well as the combination of the two hormones. E2 (10⁻¹² to 10^-8M) enhanced the differentiation of CD11b⁺CD11c⁺ DCs from BM precursor cells, and promoted the expression of CD40 and MHC Class-II, in a dose-dependent manner. In contrast, P4 (10⁻⁹ to 10^-5M) inhibited DC differentiation, but only at the highest concentrations. These effects on BMDCs were observed both in the presence or absence of LPS. When both hormones were combined, higher concentrations of P4, at levels seen in pregnancy (10^-6M) reversed the E2 effects, regardless of the concentration of E2, especially in the absence of LPS. Functionally, antigen uptake was decreased and pro-inflammatory cytokines, IL-12, IL-1 and IL-6 production by CD11b⁺CD11c⁺ DCs, was increased in the presence of E2 and these effects were reversed by high concentrations of P4. Our results demonstrate the distinct effects of E2 and P4 on differentiation and functions of bone marrow myeloid DCs. The dominating effect of higher physiological concentrations of P4 provides insight into how DC functions could be modulated during pregnancy.     

Interleukin-17 Induces an Atypical M2-Like Macrophage Subpopulation That Regulates Intestinal Inflammation

Interleukin 17 (IL-17) is a pleiotropic cytokine that acts on both immune and non-immune cells and is generally implicated in inflammatory and autoimmune diseases. Although IL-17 as well as their source, mainly but not limited to Th17 cells, is also abundant in the inflamed intestine, the role of IL-17 in inflammatory bowel disease remains controversial. In the present study, by using IL-17 knockout (KO) mice, we investigated the role of IL-17 in colitis, with special focus on the macrophage subpopulations. Here we show that IL-17KO mice had increased susceptibility to DSS-induced colitis which was associated with decrease in expression of mRNAs implicated in M2 and/or wound healing macrophages, such as IL-10, IL-1 receptor antagonist, arginase 1, cyclooxygenase 2, and indoleamine 2,3-dioxygenase. Lamina propria leukocytes from inflamed colon of IL-17KO mice contained fewer CD11b⁺Ly6C⁺MHC Class II⁺ macrophages, which were derived, at least partly, from blood monocytes, as compared to those of WT mice. FACS-purified CD11b⁺ cells from WT mice, which were more abundant in Ly6C⁺MHC Class II⁺ cells, expressed increased levels of genes associated M2/wound healing macrophages and also M1/proinflammatory macrophages. Depletion of this population by topical administration of clodronate-liposome in the colon of WT mice resulted in the exacerbation of colitis. These results demonstrate that IL-17 confers protection against the development of severe colitis through the induction of an atypical M2-like macrophage subpopulation. Our findings reveal a previously unappreciated mechanism by which IL-17 exerts a protective function in colitis.     

Monocytes Infiltrate the Pancreas via the MCP-1/CCR2 Pathway and Differentiate into Stellate Cells

Recent studies have shown that monocytes possess pluripotent plasticity. We previously reported that monocytes could differentiate into hepatic stellate cells. Although stellate cells are also present in the pancreas, their origin remains unclear. An accumulation of enhanced green fluorescent protein (EGFP)⁺CD45^– cells was observed in the pancreases and livers of chimeric mice, which were transplanted with a single hematopoietic stem cell isolated from EGFP-transgenic mice and treated with carbon tetrachloride (CCl₄). Because the vast majority of EGFP⁺CD45^– cells in the pancreas expressed stellate cell-associated antigens such as vimentin, desmin, glial fibrillary acidic protein, procollagen-I, and α-smooth muscle actin, they were characterized as pancreatic stellate cells (PaSCs). EGFP⁺ PaSCs were also observed in CCl₄-treated mice adoptively transferred with monocytes but not with other cell lineages isolated from EGFP-transgenic mice. The expression of monocyte chemoattractant protein-1 (MCP-1) and angiotensin II (Ang II) increased in the pancreas of CCl₄-treated mice and their respective receptors, C-C chemokine receptor 2 (CCR2) and Ang II type 1 receptor (AT1R), were expressed on Ly6C^high monocytes isolated from EGFP-transgenic mice. We examined the effect of an AT1R antagonist, irbesartan, which is also a CCR2 antagonist, on the migration of monocytes into the pancreas. Monocytes migrated toward MCP-1 but not Ang II in vitro. Irbesartan inhibited not only their in vitro chemotaxis but also in vivo migration of adoptively transferred monocytes from peripheral blood into the pancreas. Irbesartan treatment significantly reduced the numbers of EGFP⁺F4/80⁺CCR2⁺ monocytic cells and EGFP⁺ PaSCs in the pancreas of CCl₄-treated chimeric mice receiving EGFP⁺ bone marrow cells. A specific CCR2 antagonist RS504393 inhibited the occurrence of EGFP⁺ PaSCs in injured mice. We propose that CCR2⁺ monocytes migrate into the pancreas possibly via the MCP-1/CCR2 pathway and give rise to PaSCs.     

TNF Induction of NF-κB RelB Enhances RANKL-Induced Osteoclastogenesis by Promoting Inflammatory Macrophage Differentiation but also Limits It through Suppression of NFATc1 Expression

TNF induces bone loss in common bone diseases by promoting osteoclast formation directly and indirectly, but it also limits osteoclast formation by inducing expression of NF-κB p100. Osteoclast precursors (OCPs) are derived from M1 (inflammatory) and M2 (resident) macrophages. However, it is not known if TNF stimulates or limits osteoclast formation through regulation of M1 or M2 differentiation or if RelB, a partner of p100, is involved. To investigate these questions, we treated bone marrow cells (BMCs) with M-CSF alone or in combination with TNF to enrich for OCPs, which we called M-OCPs and T-OCPs, respectively. We found that TNF switched CD11b⁺F4/80⁺ M-OCPs from Ly6C^-Gr1^- M2 to Ly6C⁺Gr1^-CD11c⁺ and Ly6C^-Gr1^-CD11c⁺ M1 cells. RANKL induced osteoclast formation from both Ly6C⁺Gr1^- and Ly6C^-Gr1^- T-OCPs, but only from Ly6C⁺Gr1^- M-OCPs, which formed significantly fewer osteoclasts than T-OCPs. Importantly, Ly6C⁺Gr1^- cells from both M- and T-OCPs have increased expression of the M1 marker genes, iNOS, TNF, IL-1β and TGFβ1, compared to Ly6C^-Gr1^- cells, and Ly6C^-Gr1^- cells from T-OCPs also have increased expression of iNOS and TGFβ1 compared to cells from M-OCPs. Both RANKL and TNF increased RelB mRNA expression. TNF significantly increased RelB protein levels, but RANKL did not because it also induced RelB proteasomal degradation. TNF inhibited RANKL-induced NFATc1 mRNA expression and osteoclast formation from M-OCPs, but not from T-OCPs, and it did not induce Ly6C⁺Gr1^-CD11c⁺ or Ly6C^-Gr1^-CD11c⁺ M1 macrophages from RelB-/- BMCs. Furthermore, overexpression of RelB in M-OCPs reduced RANKL-induced osteoclast formation and NFATc1 mRNA expression, but it increased TNF-induced OC formation without affecting NFATc1 levels. Thus, TNF induction of RelB directly mediates terminal osteoclast differentiation independent of NFATc1 and limits RANKL-induced osteoclastogenesis by inhibiting NFATc1 activation. However, the dominant role of TNF is to expand the OCP pool by switching the differentiation of M-CSF-induced M2 to M1 macrophages with enhanced osteoclast forming potential. Strategies to degrade RelB could prevent TNF-induced M2/M1 switching and reduce osteoclast formation.     

Induction of Mouse Melioidosis with Meningitis by CD11b+ Phagocytic Cells Harboring Intracellular B. pseudomallei as a Trojan Horse

Background

Approximately 3–5% of patients with melioidosis manifest CNS symptoms; however, the clinical data regarding neurological melioidosis are limited.

Methods and Findings

We established a mouse model of melioidosis with meningitis characterized by neutrophil infiltration into the meninges histologically and B. pseudomallei in the cerebrospinal fluid (CSF) by bacteriological culturing methods. As the disease progresses, the bacteria successively colonize the spleen, liver, bone marrow (BM) and brain and invade splenic and BM cells by days 2 and 6 post-infection, respectively. The predominant cell types intracellularly infected with B. pseudomallei were splenic and BM CD11b⁺ populations. The CD11b⁺Ly6C^high inflamed monocytes, CD11b⁺Ly6C^low resident monocytes, CD11b⁺Ly6G⁺ neutrophils, CD11b⁺F4/80⁺ macrophages and CD11b⁺CD19⁺ B cells were expanded in the spleen and BM during the progression of melioidosis. After adoptive transfer of CD11b populations harboring B. pseudomallei, the infected CD11b⁺ cells induced bacterial colonization in the brain, whereas CD11b⁻ cells only partially induced colonization; extracellular (free) B. pseudomallei were unable to colonize the brain. CD62L (selectin) was absent on splenic CD11b⁺ cells on day 4 but was expressed on day 10 post-infection. Adoptive transfer of CD11b⁺ cells expressing CD62L (harvested on day 10 post-infection) resulted in meningitis in the recipients, but transfer of CD11b⁺ CD62L-negative cells did not.

Conclusions/Significance

We suggest that B. pseudomallei-infected CD11b⁺ selectin-expressing cells act as a Trojan horse and are able to transmigrate across endothelial cells, resulting in melioidosis with meningitis.     

Alternatively Activated Mononuclear Phagocytes from the Skin Site of Infection and the Impact of IL-4Rα Signalling on CD4+T Cell Survival in Draining Lymph Nodes after Repeated Exposure to Schistosoma mansoni Cercariae

In a murine model of repeated exposure of the skin to infective Schistosoma mansoni cercariae, events leading to the priming of CD4 cells in the skin draining lymph nodes were examined. The dermal exudate cell (DEC) population recovered from repeatedly (4x) exposed skin contained an influx of mononuclear phagocytes comprising three distinct populations according to their differential expression of F4/80 and MHC-II. As determined by gene expression analysis, all three DEC populations (F4/80^-MHC-II^high, F4/80⁺MHC-II^high, F4/80⁺MHC-II^int) exhibited major up-regulation of genes associated with alternative activation. The gene encoding RELMα (hallmark of alternatively activated cells) was highly up-regulated in all three DEC populations. However, in 4x infected mice deficient in RELMα, there was no change in the extent of inflammation at the skin infection site compared to 4x infected wild-type cohorts, nor was there a difference in the abundance of different mononuclear phagocyte DEC populations. The absence of RELMα resulted in greater numbers of CD4⁺ cells in the skin draining lymph nodes (sdLN) of 4x infected mice, although they remained hypo-responsive. Using mice deficient for IL-4Rα, in which alternative activation is compromised, we show that after repeated schistosome infection, levels of regulatory IL-10 in the skin were reduced, accompanied by increased numbers of MHC-II^high cells and CD4⁺ T cells in the skin. There were also increased numbers of CD4⁺ T cells in the sdLN in the absence of IL-4Rα compared to cells from singly infected mice. Although their ability to proliferate was still compromised, increased cellularity of sdLN from 4x IL-4RαKO mice correlated with reduced expression of Fas/FasL, resulting in decreased apoptosis and cell death but increased numbers of viable CD4⁺ T cells. This study highlights a mechanism through which IL-4Rα may regulate the immune system through the induction of IL-10 and regulation of Fas/FasL mediated cell death.     

CD4+ T cell-mediated cytotoxicity is associated with MHC class II expression on malignant CD19+ B cells in diffuse large B cell lymphoma

Diffuse large B cell lymphoma (DLBCL) is a common B cell malignancy with approximately 30% of patients present relapsed or refractory disease after first-line therapy. Research of further treatment options is needed. Cytotoxic CD4⁺ T cells express cytolytic molecules and have potential antitumor function. Here, we showed that the CD19⁺ cells from DLBCL patients presented significantly reduced expression of MHC II molecules than those from healthy controls. Three years after the first-line treatment, patients that presented relapsed disease had significantly lower MHC II expression on their CD19⁺ cells than patients who did not show recurrence. Examining cytotoxic CD4⁺ T cells show that DLBCL patients presented significantly elevated frequencies of granzyme A-, granzyme B-, and/or perforin-expressing cytotoxic CD4⁺ T cells. Also, frequency of cytotoxic CD4⁺ T cells in DLBCL patients was positively correlated with the MHC II expression level. Subsequently, the cytotoxic potential of CD4⁺ T cells against autologous CD19⁺ cells was investigated. We found that the cytotoxic potential of CD4⁺ T cells was highest in MHC II-high, intermediate in MHC II-mid, and lowest in MHC II-low patients. The percentage of MHC II-expressing viable CD19⁺ cells presented a significant reduction after longer incubation with cytotoxic CD4⁺ T cells, suggesting that cytotoxic CD4⁺ T cells preferentially eliminated MHC II-expressing CD19⁺ cells. Blocking MHC II on CD19⁺ cells significantly reduced the cytolytic capacity of CD4⁺ T cells. Despite these discoveries, the frequency of cytotoxic CD4⁺ T cells did not predict the clinical outcome of DLBCL patients. Together, these results demonstrated that cytotoxic CD4⁺ T cells presented an MHC II-dependent cytotoxic potential against autologous CD19⁺ cells and could potentially represent a future treatment option for DLBCL.     

Chemokine Receptor Ccr6 Deficiency Alters Hepatic Inflammatory Cell Recruitment and Promotes Liver Inflammation and Fibrosis

Chronic liver diseases are characterized by a sustained inflammatory response in which chemokines and chemokine-receptors orchestrate inflammatory cell recruitment. In this study we investigated the role of the chemokine receptor CCR6 in acute and chronic liver injury. In the absence of liver injury Ccr6 ^-/- mice presented a higher number of hepatic macrophages and increased expression of pro-inflammatory cytokines and M1 markers Tnf-α, Il6 and Mcp1. Inflammation and cell recruitment were increased after carbon tetrachloride-induced acute liver injury in Ccr6 ^-/- mice. Moreover, chronic liver injury by carbon tetrachloride in Ccr6 ^-/- mice was associated with enhanced inflammation and fibrosis, altered macrophage recruitment, enhanced CD4⁺ cells and a reduction in Th17 (CD4⁺IL17⁺) and mature dendritic (MHCII⁺CD11c⁺) cells recruitment. Clodronate depletion of macrophages in Ccr6 ^-/- mice resulted in a reduction of hepatic pro-inflammatory and pro-fibrogenic markers in the absence and after liver injury. Finally, increased CCR6 hepatic expression in patients with alcoholic hepatitis was found to correlate with liver expression of CCL20 and severity of liver disease. In conclusion, CCR6 deficiency affects hepatic inflammatory cell recruitment resulting in the promotion of hepatic inflammation and fibrosis.