Stromal cells (SCs) establish the compartmentalization of lymphoid tissues critical to the immune response. However, the full diversity of lymph node (LN) SCs remains undefined. Using droplet-based single-cell RNA sequencing, we identified nine peripheral LN non-endothelial SC clusters. Included are the established subsets, Ccl19hi T-zone reticular cells (TRCs), marginal reticular cells, follicular dendritic cells (FDCs), and perivascular cells. We also identified Ccl19lo TRCs, likely including cholesterol-25-hydroxylase+ cells located at the T-zone perimeter, Cxcl9+ TRCs in the T-zone and interfollicular region, CD34+ SCs in the capsule and medullary vessel adventitia, indolethylamine N-methyltransferase+ SCs in the medullary cords, and Nr4a1+ SCs in several niches. These data help define how transcriptionally distinct LN SCs support niche-restricted immune functions and provide evidence that many SCs are in an activated state.

A single G protein-coupled receptor (GPCR) can activate multiple signaling cascades based on the binding of different ligands. The biological relevance of this feature in immune regulation has not been evaluated. The chemokine-binding GPCR CXCR3 is preferentially expressed on CD4+ T cells, and canonically binds 3 structurally related chemokines: CXCL9, CXCL10, and CXCL11. Here we have shown that CXCL10/CXCR3 interactions drive effector Th1 polarization via STAT1, STAT4, and STAT5 phosphorylation, while CXCL11/CXCR3 binding induces an immunotolerizing state that is characterized by IL-10(hi) (Tr1) and IL-4(hi) (Th2) cells, mediated via p70 kinase/mTOR in STAT3- and STAT6-dependent pathways. CXCL11 binds CXCR3 with a higher affinity than CXCL10, suggesting that CXCL11 has the potential to restrain inflammatory autoimmunity. We generated a CXCL11-Ig fusion molecule and evaluated its use in the EAE model of inflammatory autoimmune disease. Administration of CXCL11-Ig during the first episode of relapsing EAE in SJL/J mice not only led to rapid remission, but also prevented subsequent relapse. Using GFP-expressing effector CD4+ T cells, we observed that successful therapy was associated with reduced accumulation of these cells at the autoimmune site. Finally, we showed that very low doses of CXCL11 rapidly suppress signs of EAE in C57BL/6 mice lacking functional CXCL11.

An impressive clinical success has been observed in treating a variety of cancers using immunotherapy with programmed cell death-1 (PD-1) checkpoint blockade. However, limited response in most patients treated with anti-PD-1 antibodies remains a challenge, requiring better understanding of molecular mechanisms limiting immunotherapy. In colorectal cancer (CRC) resistant to immunotherapy, mismatch-repair-proficient or microsatellite instability-low (pMMR-MSI-L) tumors have low mutation burden and constitute ~85% of patients. Here, we show that inhibition of N⁶ -methyladenosine (m⁶ A) mRNA modification by depletion of methyltransferases, Mettl3 and Mettl14, enhanced response to anti-PD-1 treatment in pMMR-MSI-L CRC and melanoma. Mettl3- or Mettl14-deficient tumors increased cytotoxic tumor-infiltrating CD8⁺ T cells and elevated secretion of IFN-γ, Cxcl9, and Cxcl10 in tumor microenvironment in vivo. Mechanistically, Mettl3 or Mettl14 loss promoted IFN-γ-Stat1-Irf1 signaling through stabilizing the Stat1 and Irf1 mRNA via Ythdf2. Finally, we found a negative correlation between METTL3 or METTL14 and STAT1 in 59 patients with pMMR-MSI-L CRC tumors. Altogether, our findings uncover a new awareness of the function of RNA methylation in adaptive immunity and provide METTL3 and METTL14 as potential therapeutic targets in anticancer immunotherapy.

Keywords: CD8+ T cells; colorectal carcinoma; immunotherapy; m6A methylation.

Psoriasis has been considered an autoimmune, T cell-mediated disorder in which adaptive immune responses predominate over those of non-antigen-specific innate immunity. To test this hypothesis, we profiled the transcriptome of psoriatic tissue and compared the data with that from cultured human keratinocytes exposed to the proinflammatory cytokine interleukin (IL)-1alpha and the Th1 cytokine interferon-gamma. When compared with patient-matched, nonlesional skin biopsies, psoriatic samples exhibited regulation of 90 transcripts including several members of the epidermal differentiation complex, molecules with antimicrobial activity, and hyperproliferation-associated keratins. Stimulation of keratinocytes with interferon-gamma resulted in regulation of 252 transcripts, with particularly strong expression of the CXCR3-binding ligands CXCL9, -10, and -11 and class II major histocompatibility complex genes, primarily those of the HLA-DR and -DP families. In contrast, the transcriptome resulting from exposure of keratinocytes to IL-1alpha elicited differences in just 19 transcripts, particularly genes within the epidermal differentiation complex and antimicrobial molecules, including PI3 and DEFB4. Major differences between the two keratinocyte transcriptomes were exhibited with only five induced IL-1alpha transcripts also regulated in the interferon-gamma set. Unexpectedly, there was a high correlation between psoriatic lesional tissue and the IL-1alpha transcriptome. These findings suggest that the inflammatory milieu in the epidermal microenvironment in psoriasis is more likely dependent on evolutionarily ancient cytokines such as IL-1, rather than those of the adaptive immune response.

Cytokine-induced inflammation is involved in the pathogenesis of type 2 diabetes mellitus (DM). We investigated plasma concentrations and ex vivo production of cytokines and chemokines, and intracellular signalling molecules, mitogen-activated protein kinases (MAPK) in T helper (Th) cells and monocytes in 94 type 2 diabetic patients with or without nephropathy and 20 healthy controls. Plasma concentrations of inflammatory cytokines tumour necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-18 and chemokine CCL2 in patients with diabetic nephropathy (DN) were significantly higher than control subjects, while IL-10, CXCL8, CXCL9, CXCL10 and adiponectin concentrations of DN were significantly higher than patients without diabetic nephropathy (NDN) and control subjects (all P < 0.05). Plasma concentrations of TNF-alpha, IL-6, IL-10, IL-18, CCL2, CXCL8, CXCL9, CXCL10 and adiponectin exhibited significant positive correlation with urine albumin : creatinine ratio in DN patients. The percentage increases of ex vivo production of IL-6, CXCL8, CXCL10, CCL2 and CCL5 upon TNF-alpha activation were significantly higher in both NDN and DN patients than controls (all P < 0.05). The percentage increases in IL-18-induced phosphorylation of extracellular signal-regulated kinase (ERK) in Th cells of NDN and DN were significantly higher than controls (P < 0.05), while the percentage increase in TNF-alpha-induced phosphorylation of p38 MAPK in monocytes and IL-18-induced phosphorylation of p38 MAPK in Th cells and monocytes were significantly higher in NDN patients than controls. These results confirmed that the aberrant production of inflammatory cytokines and chemokines and differential activation of MAPK in different leucocytes are the underlying immunopathological mechanisms of type 2 DM patients with DN.

Central nervous system (CNS) infection with herpes simplex virus (HSV)-1 triggers neuroinflammatory responses leading to peripheral immune cell infiltration into the brain. Previous in vitro studies from our laboratory, using primary human brain cells, implicated microglia as the cellular source of infection-induced chemokines, such as CXC ligand 10 (CXCL10) and CC ligand 2 (CCL2). Here, we evaluated the role of microglial cells in HSV-induced neuroimmune responses using an in vivo murine model of herpes encephalitis. Data obtained during this study demonstrated robust levels of CXCL10, CCL2 and CXCL9 detectable in the brains of infected BALB/c mice between 5 and 8 days post-infection (p.i.). Microglial cells were identified as a source of this HSV-induced chemokine production. Additional experiments established that induction of these immune mediators preceded the presence of CD3, CD4, CD8, and CD45 mRNA in the brain, and immunohistochemical analysis confirmed the presence of infiltrating CD3(+) cells. Further analysis suggested that microglia-derived chemokines drive peripheral immune cell chemotaxis, as antibodies to CXCL10 and CCL2 blocked the migration of murine splenocytes toward HSV-infected microglia by approximately 59.3+/-4.1% and 17.5+/-1.4%, respectively. Taken together, these results demonstrate that a vigorous microglia-driven cascade of pro-inflammatory immune responses is not sufficient to protect susceptible mice from HSV-1 brain infection.

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.

BACKGROUND

Obesity is a significant risk factor for psoriasis and body mass index (BMI) correlates with disease severity. Objectives To investigate the relationship between obesity and psoriasis, focusing on the role of adipokines such as leptin and resistin.

METHODS

Patients with psoriasis (n = 30) were recruited and their BMI, waist circumference and disease severity [Psoriasis Area and Severity Index (PASI)] were recorded. Fasting serum samples were obtained on enrolment and after a course of ultraviolet (UV) B treatment. Age-, sex- and BMI-matched healthy controls were also recruited.

RESULTS

On enrolment, serum leptin and soluble leptin receptor levels were not raised compared with the controls. However, resistin, interleukin (IL)-1beta, IL-6, and chemokines CCL2, CXCL8 and CXCL9 were all significantly elevated in the patient group and serum resistin correlated with disease severity (r = 0.372, P = 0.043). Improvement after UVB treatment was accompanied by decreased serum CXCL8. In vitro, both leptin and resistin could induce CXCL8 and tumour necrosis factor-alpha production by blood monocytes, and leptin could additionally induce IL-1beta and IL-1 receptor antagonist production. Leptin also dose dependently increased secretion of the growth factor amphiregulin by ex vivo-cultured lesional psoriasis skin.

CONCLUSIONS

These data support the view that leptin and resistin may be involved in the pathogenesis of psoriasis in overweight individuals, possibly by augmenting the cytokine expression by the inflammatory infiltrate.

Herpes simplex virus type 1 ocular infection elicits a potent inflammatory response including the production of the chemokines, CXCL9 and CXCL10, in mice. Since HSV-1 nucleic acid is recognized by pattern receptors including Toll-like receptor (TLR) 9, we tested the hypothesis that TLR9 is necessary for the early augmentation of CXCL10 following HSV-1 infection. Similar to wild type controls, TLR9 deficient mice constitutively expressed CXCL10 in the cornea. Following infection or stimulation with the deoxycytidylate-phosphate-deoxyguanylate (CpG) motif, CXCL10 levels were significantly elevated in the cornea of wild type but not TLR9 or type I interferon receptor deficient mice. The reduced CXCL10 response in the cornea of TLR deficient mice was correlative with an increase in virus shedding and a reduction in neutrophil infiltration. This is the first report that shows enhanced CXCL10 expression following neurotropic viral replication requires both intact TLR 9 and type I interferon signaling pathways.

Angiogenesis and inflammation are the 2 major stroma reactions in colorectal carcinoma (CRC). Guanylate binding protein-1 (GBP-1) is a key mediator of angiostatic effects of inflammation. Therefore, we hypothesized that GBP-1 may be a biomarker of intrinsic angiostasis associated with an improved outcome in CRC patients. GBP-1 was strongly expressed in endothelial cells and immune cells in the desmoplastic stroma of 32% of CRC as determined by immunohistochemical investigation of 388 sporadic CRC. Cancer-related 5-year survival was highly significant (p < 0.001) increased (16.2%) in patients with GBP-1-positive CRC. Multivariate analysis showed that GBP-1 is an independent prognostic factor indicating a reduction of the relative risk of cancer-related death by the half (p = 0.032). A comparative transcriptome analysis (22,215 probe sets) of GBP-1-positive (n = 12) and -negative (n = 12) tumors showed that particularly IFN-gamma-induced genes including the major antiangiogenic chemokines CXCL9, CXCL10 and CXCL11 were coexpressed with GBP-1. Altogether our findings indicated that GBP-1 may be a novel biomarker and an active component of a Th-1-like angiostatic immune reaction in CRC. This reaction may affect patient's response to antiangiogenic therapy and the identification of such tumors may provide a novel criterion for patient selection. Moreover, the induction of a Th-1-like angiostatic immune reaction may be a promising approach for the clinical treatment of CRC.

BACKGROUND

Ulcerative colitis is characterized by relapsing mucosal inflammation where the lesions include tissue-damaging granulocytes. In addition, T cells and natural killer (NK) cells play important pathophysiologic roles. Chemokines are a large family of peptides that play key roles in the regulation of inflammation. The CXC-chemokines, growth-related oncogene (GRO)-alpha/CXCL1 and interleukin (IL)-8/CXCL8, both recruit neutrophils and possess mitogenic properties, whereas the interferon-dependent CXC-chemokines monokine induced by gamma-interferon (MIG)/CXCL9, interferon-gamma inducible protein of 10 kD/CXCL10, and IFN-inducible T cell alpha chemoattractant/CXCL11 recruit and activate T cells and NK cells.

METHODS

The expression of CXC-chemokines was studied in eight controls and in 11 patients suffering from ulcerative colitis in the distal part of the colon, before and during topical treatment with corticosteroids. Perfusates (obtained before, after 7 days, and after 28 days of treatment) and pinch biopsies (obtained before and after 28 days of treatment) were collected by colonoscopy. The rectal release of GRO-alpha and MIG was determined by enzyme-linked immunosorbent assay (ELISA), and tissue expression of the chemokines was detected in colonic tissue by immunohistochemistry.

RESULTS

In perfusates, high levels of GRO-alpha, IL-8, and MIG were detected compared with controls (p=0.02, 0.005, and p=0.03, respectively). During treatment with corticosteroids, both GRO-alpha and MIG decreased. In clinical nonresponders, characterized by sustained inflammation, the levels of GRO-alpha and MIG remained elevated. Both epithelial cells and granulocytes, present in the submucosa, expressed GRO-alpha and MIG as detected by immunohistochemistry.

CONCLUSIONS

CXC-chemokines are likely to be important in the pathophysiology of ulcerative colitis and may become targets for novel treatment strategies. In addition, GRO-alpha may serve as a marker of disease activity.

Chronic obstructive pulmonary disease (COPD) is a progressive disease associated with a cellular inflammatory response. CD8(+) T cells are implicated in COPD pathogenesis, and their numbers significantly correlate with the degree of airflow limitation. Dendritic cells (DCs) are important sentinel immune cells, but little is known about their role in initiating and maintaining the CD8 T-cell response in COPD. To investigate the mechanisms for CD8(+) T-cell recruitment to the lung, we used resected human lung tissue to analyze chemokine receptor expression by CD8(+) T cells and chemokine production by CD1a(+) DCs. Among 11 surveyed chemokine receptors, only CC chemokine receptor (CCR5), CXC chemokine receptor (CXCR) 3, and CXCR6 correlated with COPD severity as defined by criteria from the Global Initiative for Chronic Obstructive Lung Disease. The CD8(+) T cells displayed a Tc1, CD45RA(+) effector memory phenotype. CD1a(+) DCs produced the respective ligands for CCR5 and CXCR3, CCL3 and CXCL9, and levels correlated with disease severity. CD1a(+) DCs also constitutively expressed the CXCR6 ligand, CXCL16. In conclusion, we have identified major chemokine elements that potentially mediate CD8(+) T-cell infiltration during COPD progression and demonstrated that CD1a(+) mucosal-associated DCs may sustain CD8(+) T-cell recruitment/retention. Chemokine targeting may prove to be a viable treatment approach.

Plasmodium falciparum in a subset of patients can lead to a diffuse encephalopathy known as cerebral malaria (CM). Despite treatment, mortality caused by CM can be as high as 30% while 10% of survivors of the disease may experience short- and long-term neurological complications. The pathogenesis of CM involves alterations in cytokine and chemokine expression, local inflammation, vascular injury and repair processes. These diverse factors have limited the rate of discovery of prognostic predictors of fatal CM. Identification of reliable early predictors of CM severity will enable clinicians to adjust this risk with appropriate management of CM. Recent studies revealed that elevated levels of CXCL10 expression in cerebrospinal fluid and peripheral blood plasma independently predicted severe and fatal CM. CXCR3, a promiscuous receptor of CXCL10, plays an important role in pathogenesis of mouse model of CM. In this study the role of corresponding CXCR3 ligands (CXCL11, CXCL10, CXCL9 & CXCL4) in fatal or severe CM was evaluated by comparing their levels in 16 healthy control (HC), 26 mild malaria (MM), 26 cerebral malaria survivors (CMS) and 12 non-survivors (CMNS) using enzyme linked immunosorbent assay (ELISA). Levels of CXCL4 and CXCL10 were significantly elevated in CMNS patients (p < 0.05) when compared with HC, MM and CMS. Elevated plasma levels of CXCL10 and CXCL4 were tightly associated with CM mortality. Receiver Operating Characteristic (ROC) curve analysis revealed that CXCL4 and CXCL10 can discriminate CMNS from MM (p < 0.0001) and CMS (p <0.0001) with an area under the curve (AUC)=1. These results suggest that CXCL4 and CXCL10 play a prominent role in pathogenesis of CM associated death and may be used as functional or surrogate biomarkers for predicting CM severity.

The CXCR3 chemokine receptor regulates the migration of Th1 lymphocytes and responds to three ligands: CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. We screened for potential regulation of T cell responses by matrix metalloproteinase (MMP) processing of these important chemokines. The most potent of the CXCR3 ligands, CXCL11, was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry as a substrate of the PMN-specific MMP-8, macrophage-specific MMP-12, and the general leukocyte MMP-9. The 73-amino acid residue CXCL11 is processed at both the amino and carboxyl termini to generate CXCL11-(5-73), -(5-63), and -(5-58) forms. NH2-terminal truncation results in loss of agonistic properties, as shown in calcium mobilization and chemotaxis experiments using CXCR3 transfectants and human T lymphocytes. Moreover, CXCL11-(5-73) is a CXCR3 antagonist and interestingly shows enhanced affinity to heparin. However, upon COOH-terminal truncation to position 58 there is loss of antagonist activity and heparin binding. Together this highlights an unexpected site for receptor interaction and that the carboxyl terminus is critical for glycosaminoglycan binding, an essential function for the formation of chemokine gradients in vivo. Hence, MMP activity might regulate CXCL11 tissue gradients in two ways. First, the potential of CXCL11-(5-73) to compete active CXCL11 from glycosaminoglycans might lead to the formation of an antagonistic haptotactic chemokine gradient. Second, upon further truncation, MMPs disperse the CXCL11 gradients in a novel way by proteolytic loss of a COOH-terminal GAG binding site. Hence, these results reveal potential new roles in down-regulating Th1 lymphocyte chemoattraction through MMP processing of CXCL11.

Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.

Uridine nucleotides and UDP-glucose are endogenous molecules, which are released into the extracellular environment in a lytic manner after cell damage, as well as by regulated nonlytic mechanisms. Recently, a UDP-glucose-specific G(i) protein-coupled P2Y receptor, namely P2Y(14), has been cloned. In this study, we demonstrated expression of the P2Y(14) mRNA in human lung epithelial cells and in the epithelial cell lines A549 and BEAS-2B. Evidence of functional expression of the P2Y(14) receptor in these cell lines was provided by calcium measurements after stimulation with uridine 5'-diphosphoglucose (UDP-glc). Experiments with pertussis toxin and the Ca(2+)-chelator EGTA revealed participation of pertussis toxin-sensitive G(i/o)-proteins in the mobilization of Ca(2+)-ions from intracellular stores by UDP-glc. Moreover, UDP-glc increased secretion of the potent neutrophil chemoattractant CXCL8/IL-8 in A549 and BEAS-2B cells in a pertussis toxin-sensitive manner. Moreover, reverse transcription and quantitative polymerase chain reaction revealed that UDP-glc modulated mRNA levels of IL-8/CXCL8. However, stimulation of A549 and BEAS-2B cells with UDP-glc neither modified basal nor cytokine-induced secretion of the CXC-chemokines CXCL9/MIG, CXCL10/IP-10, and CXCL11/I-TAC. In addition, UDP-glc did not affect proliferation of the two cell lines. In summary, our data provide evidence for a distinct physiologic role of P2Y(14) in the selective release of specific chemokines from human airway epithelial cells.

To elucidate the molecular mechanism of glomerular events in lupus nephritis, we performed genome-wide mRNA expression analysis of glomeruli microdissected from lupus mice. MRL/lpr mice (12-week-old) were orally given vehicle or prednisolone (10 mg/kg per day) for 4 weeks. Renal histology of MRL/lpr mice revealed mesangial proliferative glomerulonephritis with cellular infiltration of macrophages, T cells, and neutrophils. We identified 567 up-regulated genes in MRL/lpr glomeruli compared to control congenic mice. Those included complement components, adhesion molecules, chemokines and their receptors, and molecules related to antigen presentation. Over 130 genes were considered preferentially or exclusively expressed in hematopoietic cell lineages possibly reflecting leukocytes accumulation. Of note is the finding that chemokines and chemokine receptors (CCL3, CCL4, CCL5, CXCL9, CXCL10, CXCL11, CXCL16, CCR5, CXCR3, and CXCR6) that are related to T helper 1 (Th1) cells accumulation were up-regulated concomitantly with increased expression of Ebi3, a subunit of IL-27 that plays a role in Th1 predominance. These changes were accompanied by increased mRNA expression of many genes that were inducible by Th1 cytokine interferon-gamma. Prednisolone markedly attenuated glomerular lesion and leukocyte influx parallel with the reduction of enhanced gene expression. The present study shows additional evidence supporting glomerular Th1 cells accumulation and their role. Our data also provide an important resource in seeking new therapeutic targets to lupus nephritis. Supplemental table: available only at http://dx.doi.org/10.1254/jphs.FP0071337.

Natural killer (NK) cells provide in vivo control of orthopoxvirus infections in association with their expansion in the draining lymph node (LN), where they are normally very rare. The mechanism of this expansion is unclear. Herein, we determined that NK-cell depletion results in enhanced infection following footpad inoculation of cowpox virus, a natural pathogen of rodents. Following cowpox virus infection in normal mice, NK cells were greatly expanded in the draining LN, were not replicating, and displayed markers similar to splenic NK cells, suggesting specific recruitment of splenic NK cells rather than in situ proliferation. Moreover, NK-cell expansion was abrogated by prior injection of clodronate-loaded liposomes, indicating a role for subcapsular sinus macrophages. Furthermore, recruitment of transferred splenic NK cells to the draining LN was pertussis toxin-sensitive, suggesting involvement of chemokine receptors. Comprehensive analysis of chemokine mRNA expression in the draining LN following infection suggested the selective involvement of CCR2, CCR5, and/or CXCR3. Mice deficient for CCR2 or CCR5 had normal NK-cell recruitment, whereas CXCR3-deficient mice displayed a major defect, which was NK cell-intrinsic. Interestingly, both induction of transcripts for CXCR3 ligands (Cxcl9 and Cxcl10) and NK-cell recruitment required IFN-γ. These data indicate that NK-cell recruitment is mediated by subcapsular sinus macrophages, IFN-γ, and CXCR3 during orthopoxvirus infection.

Dengue viruses cause two severe diseases that alter vascular fluid barrier functions, dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). Preexisting antibodies to dengue virus disposes patients to immune-enhanced edema (DSS) or hemorrhagic (DHF) disease following infection by a discrete dengue virus serotype. Although the endothelium is the primary vascular fluid barrier, direct effects of dengue virus on endothelial cells (ECs) have not been considered primary factors in pathogenesis. Here, we show that dengue virus infection of human ECs elicits immune-enhancing EC responses. Our results suggest that rapid early dengue virus proliferation within ECs is permitted by dengue virus regulation of early, but not late, beta interferon (IFN-β) responses. The analysis of EC responses following synchronous dengue virus infection revealed the high-level induction and secretion of immune cells (T cells, B cells, and mast cells) as well as activating and recruiting cytokines BAFF (119-fold), IL-6/8 (4- to 7-fold), CXCL9/10/11 (45- to 338-fold), RANTES (724-fold), and interleukin-7 (IL-7; 128-fold). Moreover, we found that properdin factor B, an alternative pathway complement activator that directs chemotactic anaphylatoxin C3a and C5a production, was induced 34-fold. Thus, dengue virus-infected ECs evoke key inflammatory responses observed in dengue virus patients which are linked to DHF and DSS. Our findings suggest that dengue virus-infected ECs directly contribute to immune enhancement, capillary permeability, viremia, and immune targeting of the endothelium. These data implicate EC responses in dengue virus pathogenesis and further rationalize therapeutic targeting of the endothelium as a means of reducing the severity of dengue virus disease.

To achieve in situ tumor antigen uptake and presentation, intratumoral administration of ex vivo-generated, gene-modified murine bone marrow-derived dendritic cells (DC) was used in a murine lung cancer model. To attract mature host DC and activated T cells at the tumor site, the DC were transduced with an adenoviral vector expressing secondary lymphoid tissue chemokine (CCL21/SLC). Sixty percent of the mice treated with 10(6) DC-AdCCL21 intratumorally (7-10 ng/ml/10(6) cells/24 h of CCL21) at weekly intervals for 3 weeks showed complete tumor eradication, whereas only 25% of mice had complete resolution of tumors when mice were treated with fibroblasts expressing CCL21. In contrast only 12% of the mice treated with unmodified or control vector modified DC (DC-AdCV) showed complete tumor eradication. DC-AdCCL21 administration led to increases in the CD4(+), CD8(+), and CD3(+)CXCR3(+) T cells, as well as DC expressing CD11c(+) DEC205(+). CD4(+)CD25(+) T-regulatory cells infiltrating the tumors were markedly reduced after DC-AdCCL21 therapy. The tumor site cellular infiltrates were accompanied by the enhanced elaboration of granulocyte macrophage colony-stimulating factor, IFN-gamma, MIG/CXCL9, IP-10/CXCL10, and interleukin 12, but decreases in the immunosuppressive mediators transforming growth factor beta and prostaglandin E(2). DC-AdCCL21-treated tumor-bearing mice showed enhanced frequency of tumor-specific T lymphocytes secreting IFN-gamma, and tumor protective immunity was induced after DC-AdCCL21 therapy. In vivo depletion of IP-10/CXCL10, MIG/CXCL9, or IFN-gamma significantly reduced the antitumor efficacy of DC-AdCCL21. These findings provide a strong rationale for the evaluation of DC-AdCCL21 in cancer immunotherapy.

Protective immunity to pathogens depends on efficient immune responses adapted to the type of pathogen and the infected tissue. Dendritic cells (DC) play a pivotal role in directing the effector T cell response to either a protective T helper type 1 (Th1) or type 2 (Th2) phenotype. Human monocyte-derived DC can be differentiated into Th1-, Th2- or Th1/Th2-promoting DC in vitro upon activation with microbial compounds or cytokines. Host defence is highly dependent on mobile leucocytes and cell trafficking is largely mediated by the interactions of chemokines with their specific receptors expressed on the surface of leucocytes. The production of chemokines by mature effector DC remains elusive. Here we assess the differential production of both inflammatory and homeostatic chemokines by monocyte-derived mature Th1/Th2-, Th1- or Th2-promoting DC and its regulation in response to CD40 ligation, thereby mimicking local engagement with activated T cells. We show that mature Th1- and Th1/Th2-, but not Th2-promoting DC, selectively express elevated levels of the inflammatory chemokines CCL2/MCP-1, CCL3/MIP-1alpha, CCL4/MIP-1beta and CCL5/RANTES, as well as the homeostatic chemokine CCL19/MIP-3beta. CCL21/6Ckine is preferentially expressed by Th2-promoting DC. Production of the Th1-attracting chemokines, CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC, is restricted to Th1-promoting DC. In contrast, expression of Th2-associated chemokines does not strictly correlate with the Th2-promoting DC phenotype, except for CCL22/MDC, which is preferentially expressed by Th2-promoting DC. Because inflammatory chemokines and Th1-associated chemokines are constitutively expressed by mature Th1-promoting DC and CCL22/MDC is constitutively expressed by mature Th2-promoting DC, we propose a novel role for mature DC present in inflamed peripheral tissues in orchestrating the immune response by recruiting appropriate leucocyte populations to the site of pathogen entry.

Chemokines regulate lymphocyte trafficking under physiologic and pathologic conditions. In this study, we have investigated the role of CXCR3 and CXCR4 in the activation of T lymphocytes and their migration to the central nervous system (CNS) using novel mutant chemokines to antagonize CXCR3 and CXCR4 specifically. A series of truncation mutants of CXCL11, which has the highest affinity for CXCR3, were synthesized, and an antagonist, CXCL11((4-79)), was obtained. CXCL11((4-79)) strongly inhibited the migration of activated mouse T cells in response to all three high-affinity CXCR3 ligands, CXCL9, 10 and 11. CXCL12((P2G2)), while exhibiting minimal agonistic activity, potently inhibited the migration of activated mouse T cells in response to CXCL12. Interfering with the action of CXCR3 and CXCR4 with these synthetic receptor antagonists inhibited experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis and reduced the accumulation of CD4(+) T cells in the CNS. Further investigation demonstrated that CXCL12((P2G2)) inhibited the sensitization phase, whereas CXCL11((4-79)) inhibited the effector phase of the immune response. Our data suggest that simultaneous targeting of CXCR4 and CXCR3 may be of benefit in the treatment of the CNS autoimmune disease.

There are no validated biomarkers for chronic GVHD (cGVHD). We used a protein microarray and subsequent sequential enzyme-linked immunosorbent assay to compare 17 patients with treatment-refractory de novo-onset cGVHD and 18 time-matched control patients without acute or chronic GVHD to identify 5 candidate proteins that distinguished cGVHD from no cGVHD: CXCL9, IL2Rα, elafin, CD13, and BAFF. We then assessed the discriminatory value of each protein individually and in composite panels in a validation cohort (n = 109). CXCL9 was found to have the highest discriminatory value with an area under the receiver operating characteristic curve of 0.83 (95% confidence interval, 0.74-0.91). CXCL9 plasma concentrations above the median were associated with a higher frequency of cGVHD even after adjustment for other factors related to developing cGVHD including age, diagnosis, donor source, and degree of HLA matching (71% vs 20%; P < .001). A separate validation cohort from a different transplant center (n = 211) confirmed that CXCL9 plasma concentrations above the median were associated with more frequent newly diagnosed cGVHD after adjusting for the aforementioned factors (84% vs 60%; P = .001). Our results confirm that CXCL9 is elevated in patients with newly diagnosed cGVHD.

BACKGROUND

Mucosal macrophages are involved in the maintenance of epithelial barrier integrity and the elimination of invading pathogens. Although an intestinal barrier defect and microbial translocation are hallmarks of human immunodeficiency virus (HIV) infection, recent data on gut mucosal macrophages in HIV infection are sparse.

METHODS

Treatment-naive and treated HIV-infected patients and healthy controls were studied for frequencies and functional parameters of blood monocytes and macrophages in duodenal mucosa.

RESULTS

We found mucosal enrichment of macrophages in untreated HIV infection associated with reduced monocyte counts in blood and increased monocyte expression of the gut-homing molecule integrin β7. Increased CCR2 density on integrin β7-expressing monocytes and mucosal secretion of CCL2 suggest that CCR2/CCL2-chemotaxis is involved in enhanced trafficking of blood monocytes to the gut. Secretion of macrophage-related proinflammatory molecules interleukin 1β, CCL5, CXCL9, and CXCL10 was increased in the gut mucosa of untreated patients. Moreover, mucosal macrophages of untreated patients showed reduced phagocytic activity.

CONCLUSIONS

These data suggest a role for gut mucosal macrophages in HIV immune pathogenesis: infiltrated macrophages in the intestinal mucosa may promote local inflammation and tissue injury, whereas their low phagocytic activity prevents the efficient elimination of luminal antigens that cross the damaged intestinal barrier.