Recently, inhibitory cytokine pathways for leukocyte chemoattraction and activation have been identified, but there is little insight into the operational mechanisms except for models that rely on simple receptor antagonism. We have previously identified the existence of a murine eosinophil inhibitory pathway mediated by the CXC chemokine ligand 9 (CXCL9, Mig [monokine induced by interferon-gamma]) that impressively blocks eosinophil chemoattraction and function, but the mechanism has remained elusive. We now demonstrate that Mig's inhibitory action extends beyond receptor antagonism alone. Notably, in addition to inhibiting eotaxin-induced filamentous actin (F-actin) formation and chemoattraction, Mig potently blocks platelet activating factor (PAF)- and leukotriene B4 (LTB4)-induced responses. Remarkably, Mig-treated eosinophils display an abnormal F-actin assembly in the absence of agonist stimulation. Additionally, Mig pretreatment inhibits eotaxin-induced activation of the Rho-guanosine triphosphatase (GTPase) Rac, and Rac2-deficient eosinophils demonstrate an impaired transmigration and actin polymerization response to eotaxin stimulation. Furthermore, Mig was unable to inhibit eotaxin-induced responses in Rac2-deficient eosinophils. Finally, using CCR3 gene-targeted cells, Mig's inhibitory activity is demonstrated to be mediated by CC chemokine receptor 3 (CCR3). Thus, by altering agonist-induced signaling and abrogating cytoskeletal reorganization by a Rac2-dependent mechanism, Mig markedly inhibits eosinophil responses to diverse stimuli. These results establish evidence that distinct chemokines can use CCR3 to induce opposing signals in eosinophils.

Inflammatory chemokines are critical contributors in attracting relevant immune cells to the tumor microenvironment and driving cellular interactions and molecular signaling cascades that dictate the ultimate outcome of host anti-tumor immune response. Therefore, rational application of chemokines in a spatial-temporal dependent manner may constitute an attractive adjuvant in immunotherapeutic approaches against cancer. Existing data suggest that the macrophage inflammatory protein (MIP)-1 family and related proteins, consisting of CCL3 (MIP-1α), CCL4 (MIP-1β), and CCL5 (RANTES), can be major determinant of immune cellular infiltration in certain tumors through their direct recruitment of antigen presenting cells, including dendritic cells (DCs) to the tumor site. In this study, we examined how CCL3 in a murine colon tumor microenvironment, CT26, enhances antitumor immunity. We identified natural killer (NK) cells as a major lymphocyte subtype that is preferentially recruited to the CCL3-rich tumor site. NK cells contribute to the overall IFNγ content, CD103+ DC accumulation, and augment the production of chemokines CXCL9 and CXCL10 for enhanced T cell recruitment. We further demonstrate that both soluble CCL3 and CCL3-secreting irradiated tumor vaccine can effectively halt the progression of established tumors in a spatial-dependent manner. Our finding implies an important contribution of NK in the CCL3 - CD103+ DC - CXCL9/10 signaling axis in determining tumor immune landscape within the tumor microenvironment.

Metastatic colonization relies on interactions between disseminated cancer cells and the microenvironment in secondary organs. Here, we show that disseminated breast cancer cells evoke phenotypic changes in lung fibroblasts, forming a supportive metastatic niche. Colonization of the lungs confers an inflammatory phenotype in metastasis-associated fibroblasts. Specifically, IL-1α and IL-1β secreted by breast cancer cells induce CXCL9 and CXCL10 production in lung fibroblasts via NF-κB signaling, fueling the growth of lung metastases. Notably, we find that the chemokine receptor CXCR3, that binds CXCL9/10, is specifically expressed in a small subset of breast cancer cells, which exhibits tumor-initiating ability when co-transplanted with fibroblasts and has high JNK signaling that drives IL-1α/β expression. Importantly, disruption of the intercellular JNK-IL-1-CXCL9/10-CXCR3 axis reduces metastatic colonization in xenograft and syngeneic mouse models. These data mechanistically demonstrate an essential role for the molecular crosstalk between breast cancer cells and their fibroblast niche in the progression of metastasis.

Respiratory epithelial cells and macrophages are the key innate immune cells that play an important role in the pathogenesis of influenza A virus infection. We found that these two cell types from both human and pig showed comparable susceptibilities to initial infection with a highly pathogenic avian influenza (HPAI) H5N1 virus (A/turkey/Turkey/1/05) and a moderately pathogenic human influenza H1N1 virus (A/USSR/77), but there were contrasting differences in host innate immune responses. Human cells mounted vigorous cytokine (tumor necrosis factor alpha [TNF-α] and interleukin-6 [IL-6]) and chemokine (CXCL9, CXCL10, and CXCL11) responses to H5N1 virus infection. However, pig epithelial cells and macrophages showed weak or no TNF-α and chemokine induction with the same infections. The apparent lack of a strong proinflammatory response, corroborated by the absence of TNF-α induction in H5N1 virus-challenged pigs, coincided with greater cell death and the reduced release of infectious virus from infected pig epithelial cells. Suppressor of cytokine signaling 3 (SOCS3), a protein suppressor of the JAK-STAT pathway, was constitutively highly expressed and transcriptionally upregulated in H5N1 virus-infected pig epithelial cells and macrophages, in contrast to the corresponding human cells. The overexpression of SOCS3 in infected human macrophages dampened TNF-α induction. In summary, we found that the reported low susceptibility of pigs to contemporary Eurasian HPAI H5N1 virus infections coincides at the level of innate immunity of respiratory epithelial cells and macrophages with a reduced output of viable virus and an attenuated proinflammatory response, possibly mediated in part by SOCS3, which could serve as a target in the treatment or prevention of virus-induced hypercytokinemia, as observed for humans.

BACKGROUND

The pathogenesis of macrophage activation syndrome (MAS) is not clearly understood: a large body of evidence supports the involvement of mechanisms similar to those implicated in the setting of primary hemophagocytic lymphohistiocytosis.

OBJECTIVE

We sought to investigate the pathogenic role of IFN-γ and the therapeutic efficacy of IFN-γ neutralization in an animal model of MAS.

METHODS

We used an MAS model established in mice transgenic for human IL-6 (IL-6TG mice) challenged with LPS (MAS mice). Levels of IFN-γ and IFN-γ-inducible chemokines were evaluated by using real-time PCR in the liver and spleen and by means of ELISA in plasma. IFN-γ neutralization was achieved by using the anti-IFN-γ antibody XMG1.2 in vivo.

RESULTS

Mice with MAS showed a significant upregulation of the IFN-γ pathway, as demonstrated by increased mRNA levels of Ifng and higher levels of phospho-signal transducer and activator of transcription 1 in the liver and spleen and increased expression of the IFN-γ-inducible chemokines Cxcl9 and Cxcl10 in the liver and spleen, as well as in plasma. A marked increase in Il12a and Il12b expression was also found in livers and spleens of mice with MAS. In addition, mice with MAS had a significant increase in numbers of liver CD68+ macrophages. Mice with MAS treated with an anti-IFN-γ antibody showed a significant improvement in survival and body weight recovery associated with a significant amelioration of ferritin, fibrinogen, and alanine aminotransferase levels. In mice with MAS, treatment with the anti-IFN-γ antibody significantly decreased circulating levels of CXCL9, CXCL10, and downstream proinflammatory cytokines. The decrease in CXCL9 and CXCL10 levels paralleled the decrease in serum levels of proinflammatory cytokines and ferritin.

CONCLUSIONS

These results provide evidence for a pathogenic role of IFN-γ in the setting of MAS.

Cytokines play important roles as regulators of cell functions, and over the last decades a number of cytokine assays have been developed. The aim of the present study was to investigate the effects of age and gender on a large number of cytokines. Plasma samples were collected from 33 healthy blood donors. The samples were analyzed using a multiplex proximity extension assay (PEA) allowing simultaneous measurement of 92 cytokines and four technical controls. Biomarkers with less than 80% quantitative results were excluded leaving 63 cytokines that were analyzed for the effects of gender and age. The plasma level of three of the investigated biomarkers (DNER, MCP-4 and MMP-10) were found to be significantly different for the two genders (adjusted p-value<0.05), and 15 of the biomarkers (CCL11, CCL25, CDCP1, CSF-1, CXCL11, CXCL9, FGF-23, Flt3L, HGF, IL-10RB, MCP-3, MCP-4, MMP-10, OPG, VEGF-A) were significantly associated with age. This study reveals the effects of age and gender on a large number of cytokine assays. CXCL5 and TNFB were significantly higher in females, while the other markers with significant gender-dependent differences were higher in males. For the markers that were significantly associated with age, only CXCL6 was found to decrease with age, while the other biomarkers increased with age.

Background: Immunosuppressive and anti-cytokine treatment may have a protective effect for patients with COVID-19. Understanding the immune cell states shared between COVID-19 and other inflammatory diseases with established therapies may help nominate immunomodulatory therapies.

Methods: To identify cellular phenotypes that may be shared across tissues affected by disparate inflammatory diseases, we developed a meta-analysis and integration pipeline that models and removes the effects of technology, tissue of origin, and donor that confound cell-type identification. Using this approach, we integrated > 300,000 single-cell transcriptomic profiles from COVID-19-affected lungs and tissues from healthy subjects and patients with five inflammatory diseases: rheumatoid arthritis (RA), Crohn's disease (CD), ulcerative colitis (UC), systemic lupus erythematosus (SLE), and interstitial lung disease. We tested the association of shared immune states with severe/inflamed status compared to healthy control using mixed-effects modeling. To define environmental factors within these tissues that shape shared macrophage phenotypes, we stimulated human blood-derived macrophages with defined combinations of inflammatory factors, emphasizing in particular antiviral interferons IFN-beta (IFN-β) and IFN-gamma (IFN-γ), and pro-inflammatory cytokines such as TNF.

Results: We built an immune cell reference consisting of > 300,000 single-cell profiles from 125 healthy or disease-affected donors from COVID-19 and five inflammatory diseases. We observed a CXCL10+ CCL2+ inflammatory macrophage state that is shared and strikingly abundant in severe COVID-19 bronchoalveolar lavage samples, inflamed RA synovium, inflamed CD ileum, and UC colon. These cells exhibited a distinct arrangement of pro-inflammatory and interferon response genes, including elevated levels of CXCL10, CXCL9, CCL2, CCL3, GBP1, STAT1, and IL1B. Further, we found this macrophage phenotype is induced upon co-stimulation by IFN-γ and TNF-α.

Conclusions: Our integrative analysis identified immune cell states shared across inflamed tissues affected by inflammatory diseases and COVID-19. Our study supports a key role for IFN-γ together with TNF-α in driving an abundant inflammatory macrophage phenotype in severe COVID-19-affected lungs, as well as inflamed RA synovium, CD ileum, and UC colon, which may be targeted by existing immunomodulatory therapies.

Keywords: COVID-19; Inflammatory diseases; Macrophage heterogeneity; Macrophage stimulation; Single-cell multi-disease tissue integration; Single-cell transcriptomics.

Interferon γ (IFNγ) is considered a seminal cytokine in the pathogenesis of giant cell arteritis (GCA), but its functional role has not been investigated. We explored changes in infiltrating cells and biomarkers elicited by blocking IFNγ with a neutralising monoclonal antibody, A6, in temporal arteries from patients with GCA.

Temporal arteries from 34 patients with GCA (positive histology) and 21 controls were cultured on 3D matrix (Matrigel) and exposed to A6 or recombinant IFNγ. Changes in gene/protein expression were measured by qRT-PCR/western blot or immunoassay. Changes in infiltrating cells were assessed by immunohistochemistry/immunofluorescence. Chemotaxis/adhesion assays were performed with temporal artery-derived vascular smooth muscle cells (VSMCs) and peripheral blood mononuclear cells (PBMCs).

Blocking endogenous IFNγ with A6 abrogated STAT-1 phosphorylation in cultured GCA arteries. Furthermore, selective reduction in CXCL9, CXCL10 and CXCL11 chemokine expression was observed along with reduction in infiltrating CD68 macrophages. Adding IFNγ elicited consistent opposite effects. IFNγ induced CXCL9, CXCL10, CXCL11, CCL2 and intracellular adhesion molecule-1 expression by cultured VSMC, resulting in increased PBMC chemotaxis/adhesion. Spontaneous expression of chemokines was higher in VSMC isolated from GCA-involved arteries than in those obtained from controls. Incubation of IFNγ-treated control arteries with PBMC resulted in adhesion/infiltration by CD68 macrophages, which did not occur in untreated arteries.

Our ex vivo system suggests that IFNγ may play an important role in the recruitment of macrophages in GCA by inducing production of specific chemokines and adhesion molecules. Vascular wall components (ie, VSMC) are mediators of these functions and may facilitate progression of inflammatory infiltrates through the vessel wall.

BACKGROUND

Although antibody mechanisms play a pathogenetic role in liver allograft rejection, no data exist on B lymphocytes, plasma cells, complement, and chemokines in rejected liver tissue.

METHODS

Liver biopsy specimens from 25 patients with acute allograft rejection (AR) (rejection activity index, RAI score: 1-9) were analyzed by immunohistochemistry (IH) and reverse transcriptase-polymerase chain reaction (RT-PCR) and compared with biopsy specimens taken prior to implantation (PI). The number of CD20 and CD138 cells was evaluated, and the presence and abundance of the chemokines macrophage inflammatory protein (MIP)-3alpha, CXCL9, CXCL10, CXCL11, CXCL12, and their receptors CCR-6, CXCR3, and CXCR4 were examined. Complement depositions were visualized by C4d IH.

RESULTS

The numbers of B lymphocytes (P=0.002) and plasma cells (P=0.022) were significantly higher in AR biopsy specimens compared with PI biopsy specimens. MIP-3alpha and CCR-6 cells were detected in the portal fields of all AR biopsy specimens. IH double staining revealed a colocalization of MIP-3alpha/CD20 cells; C4d deposits could be demonstrated along the portal capillaries. All examined chemokines and receptors could be detected in normal liver tissue and in AR biopsy specimens by RT-PCR and semiquantitative RT-PCR, demonstrating an overexpression of CXCL10 and -11.

CONCLUSIONS

The significant increase of B lymphocytes and plasma cells during acute rejection, together with the lack of a significant increase of proliferating cells, indicates that the migration of B lymphocytes and plasma cells-promoted by the expression of B-cell activating chemokines/receptors-plays a key role in acute liver rejection. The C4d deposits along the portal capillaries indicate a humorally mediated alloresponse caused by the accumulated B and plasma cells.

The chemokine receptor CXCR3 is predominantly expressed on activated T and natural killer (NK) cells. CXCR3 and its ligands, CXCL11, CXCL10, and CXCL9, play a major role in T-helper 1 (Th1)-dependent inflammatory responses. CXCL11 is the most dominant physiological inducer of adhesion, migration, and internalization of CXCR3. To study the role of CXCR3 carboxyl-terminus and the third intracellular (3i) loop in chemokine-mediated migration, adhesion, and CXCR3 internalization, we generated CXCR3 receptors mutated in their distal (Ser-Thr domain) or proximal (trileucine domain) membrane carboxyl terminus, and/or the third intracellular loop. We found that migration of CXCR3-expressing HEK 293 cells toward CXCL11 was pertussis toxin-dependent and required the membrane proximal carboxyl terminus of CXCR3. Internalization induced by CXCL11 and protein kinase C (PKC) activation was also regulated by the membrane proximal carboxyl terminus; however, only CXCL11-induced internalization required the LLL motif of this region. Internalization and Ca(2+) flux induced by CXCL11 were independent of the 3i loop S245, whereas migration at high CXCL11 concentrations, integrin-dependent adhesion, and actin polymerization were S245 dependent. Our findings indicate that CXCL11-dependent CXCR3 internalization and cell migration are regulated by the CXCR3 membrane proximal carboxyl terminus, whereas adhesion is regulated by the 3i loop S245. Thus, distinct conformational changes induced by a given CXCR3 ligand trigger different downstream effectors of adhesion, motility, and CXCR3 desensitization.

Besides their antiviral activity, type I Interferons (IFN) display context-specific immunomodulation. In contrast to long-known IFNα/β, Interleukin (IL)-22 is an anti-bacterial, largely tissue protective cytokine that recently gained attention. Herein, cellular IFNα/IL-22 interactions are investigated. We report that pre-conditioning of epithelial cells with IFNα initiated dramatic changes in IL-22 signaling normally dominated by signal transducer and activator of transcription (STAT)-3. Specifically, by using human DLD1 colon epithelial/carcinoma cells we demonstrate that, upon IFNα, IL-22 converts into a cytokine robustly activating STAT1 and its downstream pro-inflammatory targets CXCL9, CXCL10, and inducible nitric oxide synthase (iNOS). Accordingly, only after IFNα pre-incubation was IL-22-induced STAT1 binding to the CXCL10 promoter detectable. Using the viral mimic polyinosinic:polycytidylic acid and the IFNα/β antagonist B18R we furthermore demonstrate the capability of endogenous IFN to promote IL-22-induced STAT1 activation and expression of CXCL10. IL-22-induced STAT1 activation subsequent to IFNα priming became likewise apparent in human Caco2 colon epithelial/carcinoma cells, HepG2 hepatoma cells, and primary keratinocytes. Current observations may relate to characteristics of IFNα/β in clinical therapy and expose margins of tissue protection by IL-22 application.

OBJECTIVE

To explore the expression of thymic stromal lymphopoietin (TSLP) in patients with diffuse cutaneous systemic sclerosis (dcSSc) and compare its effects in vivo and in vitro with those of interleukin-13 (IL-13) and transforming growth factor β (TGFβ).

METHODS

Skin biopsy specimens from patients with dcSSc (n = 14) and healthy controls (n = 13) were analyzed by immunohistochemistry and immunofluorescence for TSLP, TSLP receptor, CD4, CD8, CD31, and CD163 markers. Wild-type, IL-4Rα1-, and TSLP-deficient mice were treated with TGFβ, IL-13, poly(I-C), or TSLP by osmotic pump. Human fibroblasts and peripheral blood mononuclear cells (PBMCs) were stimulated with TGFβ, IL-13, poly(I-C), or TSLP. Microarray analysis and quantitative polymerase chain reaction were performed to determine gene expression, and protein levels of phospho-Smad2 and macrophage marker CD163 were tested.

RESULTS

TSLP was highly expressed in the skin of dcSSc patients, more strongly in perivascular areas and in immune cells, and was produced mainly by CD163+ cells. The skin of TSLP-treated mice showed up-regulated clusters of gene expression that overlapped strongly with those in IL-13- and TGFβ-treated mice. TSLP up-regulated specific genes, including CXCL9, proteasome, and interferon (IFN)-regulated genes. TSLP treatment in IL-4Rα1-deficient mice promoted similar cutaneous inflammation as in wild-type mice, though TSLP-induced arginase 1, CCL2, and matrix metalloproteinase 12 messenger RNA levels were blocked. In PBMCs, TSLP up-regulated tumor necrosis factor α, Mx-1, IFNγ, CXCL9, and mannose receptor 1 gene expression. TSLP-deficient mice treated with TGFβ showed less fibrosis and blocked expression of plasminogen activator inhibitor 1 and osteopontin 1. Poly(I-C)-treated mice showed high levels of cutaneous TSLP.

CONCLUSIONS

TSLP is highly expressed in the skin of dcSSc patients and interacts in a complex manner with 2 other profibrotic cytokines, TGFβ and IL-13, strongly suggesting that it might promote SSc fibrosis directly or indirectly by synergistically stimulating profibrotic genes, or production of these cytokines.

The selective CXC chemokine receptor 3 (CXCR3) agonists, monokine induced by interferon-gamma (IFN- gamma)/CXC chemokine ligand 9 (CXCL9), IFN-inducible protein 10/CXCL10, and IFN-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, attract CXCR3+ cells such as CD45RO+ T lymphocytes, B cells, and natural killer cells. Further, all three chemokines are potent, natural antagonists for chemokine receptor 3 (CCR3) and feature defensin-like, antimicrobial activities. In this study, we show that I-TAC, in addition to these effects, acts as an antagonist for CCR5. I-TAC inhibited the binding of macrophage-inflammatory protein-1alpha (MIP-1alpha)/CC chemokine ligand 3 (CCL3) to cells transfected with CCR5 and to monocytes. Furthermore, cell migration evoked by regulated on activation, normal T expressed and secreted (RANTES)/CCL5 and MIP-1beta/CCL4, the selective agonist of CCR5, was inhibited in transfected cells and monocytes, respectively. In two other functional assays, namely the release of free intracellular calcium and actin polymerization, I-TAC reduced CCR5 activities to minimal levels. Sequence and structure analyses indicate a potential role for K17, K49, and Q51 of I-TAC in CCR5 binding. Our results expand on the potential role of I-TAC as a negative modulator in leukocyte migration and activation, as I-TAC would specifically counteract the responses mediated by many "classical," inflammatory chemokines that act not only via CCR3 but via CCR5 as well.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH/D family proteins, and plays an important role in antiviral response via interferon-stimulated genes (ISGs) and type 1 IFN. In this study, the roles of RIG-I in the epithelial cells in the cross-talk between type 2 IFN and inducible chemokines production are high-lighted. The results showed that RIG-I was constitutively expressed in normal surface epithelia lining the colonic mucosa. RIG-I was constitutively expressed in the epithelial cell lines HT-29, and IFN-gamma and TNF-alpha enhanced the RIG-I expression in a dose-dependent manner. IFN-gamma was shown to stimulate CXCL9-11 production, and RNA interference against RIG-I resulted in significant decrease of IFN-gamma-induced CXCL9-11 productions. These results suggest that RIG-I play an important role in the cross-talk between inflammatory cytokines and immune cell trafficking. In conclusion, RIG-I might regulate the gut barrier function in homeostatic and inflammatory conditions.

Pulmonary infections and dysfunction are frequent outcomes during the development of immunodeficiency associated with human immunodeficiency virus type 1 (HIV-1) infection, and obtaining a better understanding of the immunologic changes that occur in lungs following HIV-1 infection will provide a foundation for the development of further intervention strategies. We sought here to identify changes in the pulmonary immune environment that arise during simian immunodeficiency virus (SIV) infection of rhesus macaques, which serves as an excellent model system for HIV-1 infection and disease. To examine the gene expression profiles of macaque lung tissues following infection with the pathogenic SIV/DeltaB670 isolate, we performed cDNA microarray hybridizations with lung total RNAs using two commercially available cDNA arrays and a custom-fabricated, immunologically focused macaque cDNA microarray. In situ hybridization and real-time RT-PCR were performed to provide additional analyses of gene expression. Among the genes exhibiting the highest level of induction in lung tissues were the IFN-gamma-inducible chemokines, CXCL10/IP-10 and CXCL9/Mig. In situ hybridization and real-time RT-PCR strongly supported these findings. Correlation analyses revealed that the levels of expression of IFN-gamma, CXCL9/Mig, and CXCL10/IP-10 mRNAs were all strongly positively correlated, and that CXCL10/IP-10 mRNA and Pneumocystis carinii rRNA were positively correlated. Taken together, these findings demonstrate that inflammatory chemokines are among the most differentially expressed mRNAs in macaque lung tissues during systemic SIV infection of rhesus macaques, and provide insight into the complicated events occurring in the lung tissues during HIV-1 infection in humans.

The migration of dendritic cells (DCs) to lymph nodes (LNs) is pivotal to the establishment of the immune response. DCs have been proved to pass through the afferent lymphatic pathway to enter LNs from the peripheral tissues after they have scanned for self or nonself antigens. In response to danger signals, both myeloid and plasmacytoid DC precursors (mDC and pDC precursors) are rapidly mobilized into the circulation. mDC precursors are recruited to inflamed tissues in response to inflammatory chemokines and then remobilized to regional LNs in response to CCL21. In contrast, pDC precursors directly transmigrate to regional LNs via high endothelial venules in a CXCL9- and E-selectin-dependent manner. Such migration is largely dependent on systemic inflammatory reactions. After accumulating in the LNs through distinct trafficking pathways, DCs interact with lymphocytes temporally and spatially to establish effective immune responses. The inflammation-dependent, chemokine-driven property of DC precursor trafficking is a very sophisticated host defense system.

OBJECTIVE

To investigate the effects of T-cell-derived cytokines on gene and protein expression of chemokines in a human RPE cell line (ARPE-19).

METHODS

We used an in vitro coculture system in which the RPE and CD3/CD28-activated T-cells were separated by a membrane. RPE cell expression of chemokine genes was quantified using three different types of microarrays. Protein expression was determined by single and multiplex ELISA and immunoblotting.

RESULTS

Coculture with activated T-cells increased RPE mRNA and protein expression of chemokines CCL2 (MCP-1); CCL5 (RANTES); CCL7 (MCP-3); CCL8 (MCP-2); CXCL1 (GRO-α); IL8 (CXCL8); CXCL9 (MIG); CXCL10 (IP10); CXCL11 (ITAC); and CX3CL1 (fractalkine). CCL7, CXCL9, CXCL10, and CXCL11 were secreted significantly more in the apical direction. Using recombinant human cytokines and neutralizing antibodies, we identified IFNγ and TNFα as the two major T-cell-derived cytokines responsible for the RPE response. For CCL5, CXCL9, CXCL10, CXCL11, CXCL16, and CX3CL1, we observed a synergistic effect of IFNγ and TNFα in combination. CCL20, CXCL1, CXCL6, and IL8 were negatively regulated by IFNγ.

CONCLUSIONS

RPE cells responded to exposure to T-cell-derived cytokines by upregulating expression of multiple chemokines related to microglial, T-cell, and monocyte chemotaxis and activation. This inflammatory stress response may have implications for immune homeostasis in the retina, and for the further understanding of inflammatory ocular diseases such as uveitis and AMD.

OBJECTIVE

We evaluated the utility of chimeric γc homeostatic cytokine, IL-7/IL-7Rα-Fc, to restore host APC (antigen presenting cell) and T cell activities in lung cancer.

METHODS

Utilizing murine lung cancer models we determined the antitumor efficacy of IL-7/IL-7Rα-Fc. APC, T cell, cytokine analyses, neutralization of CXCL9, CXCL10, and IFNγ were carried out to evaluate the mechanistic differences in the antitumor activity of IL-7/IL-7Rα-Fc in comparison to controls.

RESULTS

IL-7/IL-7Rα-Fc administration inhibited tumor growth and increased survival in lung cancer. Accompanying the tumor growth inhibition were increases in APC and T cell activities. In comparison to controls, IL-7/IL-7Rα-Fc treatment of tumor bearing mice led to increased: (i) levels of CXCL9, CXCL10, IFNγ, IL-12 but reduced IL-10 and TGFβ, (ii) tumor macrophage infiltrates characteristic of M1 phenotype with increased IL-12, iNOS but reduced IL-10 and arginase, (iii) frequencies of T and NK cells, (iv) T cell activation markers CXCR3, CD69 and CD127(low), (v) effector memory T cells, and (vi) T cell cytolytic activity against parental tumor cells. IL-7/IL-7Rα-Fc treatment abrogated the tumor induced reduction in splenic functional APC activity to T responder cells. The CXCR3 ligands played an important role in IL-7/IL-7Rα-Fc-mediated antitumor activity. Neutralization of CXCL9, CXCL10, or IFNγ reduced CXCR3 expressing activated T cells infiltrating the tumor and abrogated IL-7/IL-7Rα-Fc-mediated tumor growth inhibition.

CONCLUSIONS

Our findings show that IL-7/IL-7Rα-Fc promotes afferent and efferent antitumor responses in lung cancer.

Keratinocytes in the skin play an important role in innate immune responses by secreting chemokines. This study aimed to determine if keratinocyte cell lines can be used for studies of innate immune mechanisms. Human primary keratinocytes and the HaCaT, CCD 1106 KERTr (KERTr) and HEK001 cell lines were treated with a panel of Toll-like receptor (TLR)-ligands. Expression of IL-8, CCL20, CXCL9 and CXCL10 was determined. All three cell lines expressed TLR1-6 and TLR9. KERTr cells responded to the same TLR-ligands as primary keratinocytes. Overall HEK001 responded similarly, but appeared to be relatively more sensitive to flagellin. This was in agreement with increased expression of TLR5. The expression profiles were most distinct in HaCaT cells. Furthermore, our data confirm and extend previously reported TLR7 and TLR8 independent IL-8 secretion by keratinocytes after Imiquimod treatment. The different cell lines represent complementary tools for molecular studies of innate immunity of the skin.

In animal models of experimental cerebral malaria (ECM), neuropathology is associated with an overwhelming inflammatory response and sequestration of leukocytes and parasite-infected RBCs in the brain. In this study, we explored the effect of vitamin D (VD; cholecalciferol) treatment on host immunity and outcome of ECM in C57BL/6 mice during Plasmodium berghei ANKA (PbA) infection. We observed that oral administration of VD both before and after PbA infection completely protected mice from ECM. VD administration significantly dampened the inducible systemic inflammatory responses with reduced circulating cytokines IFN-γ and TNF and decreased expression of these cytokines by the spleen cells. Meanwhile, VD also resulted in decreased expression of the chemokines CXCL9 and CXCL10 and cytoadhesion molecules (ICAM-1, VCAM-1, and CD36) in the brain, leading to reduced accumulation of pathogenic T cells in the brain and ultimately substantial improvement of the blood-brain barriers of PbA-infected mice. In addition, VD inhibited the differentiation, activation, and maturation of splenic dendritic cells. Meanwhile, regulatory T cells and IL-10 expression levels were upregulated upon VD treatment. These data collectively demonstrated the suppressive function of VD on host inflammatory responses, which provides significant survival benefits in the murine ECM model.

Ketotifen is a mast cell stabilizer and useful in younger children with allergic diseases such as asthma and allergic rhinitis. Macrophage-derived chemokine (MDC) is a T-helper cell type 2 (Th2)-related chemokine involved in recruitment of Th2 cells toward allergen-challenged inflammation. However, the Th1-related chemokines, interferon-inducible protein 10 (IP-10)/CXCL10, and the monokine induced by interferon-gamma (MIG)/CXCL9 are also important in allergen-induced asthma in animal models. We investigated the effects of ketotifen on the expression of Th1- and Th2-related chemokines of human monocytes in vitro and ex vivo. Ketotifen (5-50 microM) significantly down-regulated lipopolysaccharide (LPS)-induced MDC, MIG and IP-10 (p < 0.05, each comparison) in THP-1 cells and human primary monocytes in a dose-dependent manner. SB203580 [p38-mitogen-activated protein kinase (MAPK) inhibitor] suppressed LPS-induced MDC and IP-10 expression, and PD98059 (ERK-MAPK inhibitor) could only suppress LPS-induced IP-10, but not MDC expression. LPS-induced pp38 and p-ERK expression of THP-1 monocytic cells was suppressed by ketotifen. These data demonstrate that ketotifen is effective in down-regulating LPS-induced MDC, MIG and IP-10, which play important roles in the pathogenesis of airway inflammation. The suppressive effect on MDC and IP-10 may, at least in part, involve the down-regulation of LPS-induced p38 and ERK-MAPK expression.

BACKGROUND

The chemotactic signals regulating cell trafficking in the herpes simplex virus type 1 (HSV-1) infected cornea are well documented, however, those in the cornea-associated tissues, such as the trigeminal ganglion (TG) and draining lymph nodes (LNs), are largely unknown.

OBJECTIVE

To examine chemokine expression and subsequent cell infiltration in the HSV-1 infected cornea and its associated tissues.

METHODS

Eight-week-old female BALB/c mice were infected with 10 mu l HSV-1 (CHR3 strain: 5 x 106 PFU/ml) by corneal scarification. Total RNAs were extracted from the corneas, TGs, and LNs at pre-inoculation, 3 days post-inoculation (P.I.) and 7 days P.I. The mRNA for 28 different chemokines in the extracts was amplified by RT-PCR. Infiltrating cells were identified by immunohistochemistry.

RESULTS

After the HSV-1 infection, the corneal stroma became edematous by infiltrated cells under the eroded epithelium. The TG and LNs were markedly swollen. The cornea was infiltrated with granulocytes and CD11b+ cells at 3 days P.I., followed by CD4+ and CD8+ T cells at 12 days P.I. In the TG, CD11b+ cells, but no granulocytes, infiltrated throughout the observation period. T cells migrated into the TG earlier than into the cornea. Gene expressions of neutrophil-attracting chemokines (CXCL1, 2, 3, and 5) increased in the cornea, but they did not enhance in the TG or LNs. On the other hand, gene expressions of chemokines which attract CD11b+ cells such as CCL2, 8, 7, 12, CCL3, 4, and CCL5, increased in the cornea and TG with its peak at 3 days P.I. Gene expressions of chemokines those work on T cells and B cells, such as CCL19, CCL21, CXCL9, CXCL13, CXCL10, XCL1, and CXCL16, were up-regulated and peaked at 3 days P.I. in the cornea and in the TG. Thus, pattern of chemokine gene expression was similar in the cornea and in the TG. On the contrary, gene expressions of chemokines in the draining LNs affecting CD11b+ cells and T cells were temporarily down-regulated.

CONCLUSIONS

Upon HSV-1 infection, dynamic gene expression of chemokines was observed not only in the inoculated cornea but also in its associated tissues.

BACKGROUND

The role of Mycobacteria in the etiology of Crohn's disease (CD) has been a contentious subject for many years. Recently, our laboratory showed that spontaneous colitis in IL-10-/- mice is driven in part by antigens (Ags) conserved in Mycobacteria. The present study dissects some of the common cellular and molecular mechanism that drive Mycobacteria-mediated and spontaneous colitis in IL-10-/- mice.

RESULTS

We show that serum from inflammatory bowel disease (IBD) patients contain significantly higher levels of Mycobacterium avium paratuberculosis-specific IgG1 and IgG2 antibodies (Abs), serum amyloid A (SAA) as well as CXCR3 ligands than serum from healthy donors. To study the cellular mechanisms of Mycobacteria-associated colitis, pathogen-free IL-10-/- mice were given heat-killed or live M. avium paratuberculosis. The numbers of mucosal T cells, neutrophils, NK/NKT cells that expressed TNFalpha, IFN-gamma, and/or CXCL10 were significantly higher in mice that received live Mycobacteria than other groups. The numbers of mucosal CXCR3+, CXCL9+, CXCL11+ and/or IFN-gamma+ dendritic cells (DCs) were also significantly higher in M. avium paratuberculosis-challenged mice, than compared to control mice.

CONCLUSIONS

The present study shows that CD and UC patients mount significant Mycobacteria-specific IgG1>> IgG2 and CXCR3 ligand responses. Several cellular mechanisms that drive spontaneous colitis also mediate Mycobacteria-enhanced colitis in IL-10-/- mice. Similar to IL-10-/- mice under conventional housing, we show that Mycobacteria-challenge IL-10-/- mice housed under otherwise pathogen-free conditions develop colitis that is driven by CXCR3- and CXCR3 ligand-expressing leukocytes, which underscores another important hallmark and molecular mechanism of colitis. Together, the data show that Mycobacteria-dependent host responses, namely CXCL10+ T cells and NK cells, assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes to enhance colitis of susceptible hosts.

Recently, differences in the levels of various chemokines and cytokines were reported in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) as compared with controls. Moreover, the analyte profile differed between chronic ME/CFS patients of long duration versus patients with disease of less than 3years. In the current study, we measured the plasma levels of 34 cytokines, chemokines and growth factors in 100 chronic ME/CFS patients of long duration and in 79 gender and age-matched controls. We observed highly significant reductions in the concentration of circulating interleukin (IL)-16, IL-7, and Vascular Endothelial Growth Factor A (VEGF-A) in ME/CFS patients. All three biomarkers were significantly correlated in a multivariate cluster analysis. In addition, we identified significant reductions in the concentrations of fractalkine (CX3CL1) and monokine-induced-by-IFN-γ (MIG; CXCL9) along with increases in the concentrations of eotaxin 2 (CCL24) in ME/CFS patients. Our data recapitulates previous data from another USA ME/CFS cohort in which circulating levels of IL-7 were reduced. Also, a reduced level of VEGF-A was reported previously in sera of patients with Gulf War Illness as well as in cerebral spinal fluid samples from a different cohort of USA ME/CFS patients. To our knowledge, we are the first to test for levels of IL-16 in ME/CFS patients. In combination with previous data, our work suggests that the clustered reduction of IL-7, IL-16 and VEGF-A may have physiological relevance to ME/CFS disease. This profile is ME/CFS-specific since measurement of the same analytes present in chronic infectious and autoimmune liver diseases, where persistent fatigue is also a major symptom, failed to demonstrate the same changes. Further studies of other ME/CFS and overlapping disease cohorts are warranted in future.