Mucositis arising from cancer chemotherapy is a common problem for which there is no definitive treatment. 5-fluorouracil (5-FU) is a common cytotoxic agent used to treat solid tumors. A global gene expression array was performed to identify genetic signals involved in the pathogenesis of mucositis. The chemokine (C-X-C motif) ligand 9 (CXCL9) was one of the candidates identified that presented a characteristic gene expression profile; its temporal expression pattern was correlated with the damage and regeneration phases of the small intestine upon 5-FU chemotherapy. We found that prophylactic CXCL9 administration was able to attenuate the severity of mucositis, weight loss and diarrhea caused by chemotherapy. CXCL9 also increased the tolerance of the mice to lethal-dose chemotherapy. Moreover, we demonstrated that CXCL9 was able to promote the proliferation and regeneration of intestinal cells by inhibiting the proliferation of normal intestinal mucosal cells prior to chemotherapy and by reducing the 5-FU-induced apoptosis in intestinal crypts. Thus, pretreatment with CXCL9 is a new and promising strategy for the alleviation of chemotherapy-induced intestinal mucositis in clinical settings.

BACKGROUND

Human T-lymphotropic virus type 1 (HTLV-1) -associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a rare chronic neuroinflammatory disease. Since the disease course of HAM/TSP varies among patients, there is a dire need for biomarkers capable of predicting the rate of disease progression. However, there have been no studies to date that have compared the prognostic values of multiple potential biomarkers for HAM/TSP.

RESULTS

Peripheral blood and cerebrospinal fluid (CSF) samples from HAM/TSP patients and HTLV-1-infected control subjects were obtained and tested retrospectively for several potential biomarkers, including chemokines and other cytokines, and nine optimal candidates were selected based on receiver operating characteristic (ROC) analysis. Next, we evaluated the relationship between these candidates and the rate of disease progression in HAM/TSP patients, beginning with a first cohort of 30 patients (Training Set) and proceeding to a second cohort of 23 patients (Test Set). We defined "deteriorating HAM/TSP" as distinctly worsening function (≥3 grades on Osame's Motor Disability Score (OMDS)) over four years and "stable HAM/TSP" as unchanged or only slightly worsened function (1 grade on OMDS) over four years, and we compared the levels of the candidate biomarkers in patients divided into these two groups. The CSF levels of chemokine (C-X-C motif) ligand 10 (CXCL10), CXCL9, and neopterin were well-correlated with disease progression, better even than HTLV-1 proviral load in PBMCs. Importantly, these results were validated using the Test Set.

CONCLUSIONS

As the CSF levels of CXCL10, CXCL9, and neopterin were the most strongly correlated with rate of disease progression, they represent the most viable candidates for HAM/TSP prognostic biomarkers. The identification of effective prognostic biomarkers could lead to earlier detection of high-risk patients, more patient-specific treatment options, and more productive clinical trials.

Clinical studies indicate a role of allogeneic hematopoietic cell transplantation (alloHCT) for patients with refractory or recurrent B-cell lymphoma (BCL) indicative of a graft-versus-tumor effect. However, the relevance of local immunosuppression in the BCL microenvironment by donor-derived regulatory T cells (Treg) after alloHCT is unclear. Therefore, we studied Treg recruitment after alloHCT in different murine BCL models and the impact of lymphoma-derived chemoattractive signals. Luciferase transgenic Tregs accumulated in murine BCL microenvironment and microarray-based analysis of BCL tissues revealed increased expression of CXCL9, CXCL10, and CXCL12. In vivo blocking identified the CXCR4/CXCL12 axis as being critical for Treg attraction toward BCL. In contrast to Tregs, effector T cells displayed low levels of CXCR4 and were not affected by the pharmacologic blockade. Most important, blocking CXCR4 not only reduced Treg migration toward tumor tissue but also enhanced antitumor responses after alloHCT. CXCL12 production was dependent on antigen-presenting cells (APC) located in the lymphoma microenvironment, and their diphtheria-toxin receptor (DTR)-based depletion in CD11c.DTR-Tg mice significantly reduced Treg accumulation within BCL tissue. CXCL12 was also detected in human diffuse, large BCL tissues indicative of its potential clinical relevance. In conclusion, we demonstrate that Tregs are recruited toward BCL after alloHCT by infiltrating host APCs in a CXCL12-dependent fashion. Blocking CXCR4 enhanced antitumor effects and prolonged survival of tumor-bearing mice by reducing local Treg accumulation, indicating that CXCR4 is a potential target to interfere with tumor escape after alloHCT.

Aqueous humor (AH) samples from patients with Behçet's disease (BD) (n=29), Vogt-Koyanagi-Harada (VKH) disease (n=21), and HLA-B27-associated uveitis (n=8), and 42 control patients were assayed for the neutrophil chemoattractants CXCL1/GRO-α and CXCL8/IL-8 and the lymphocyte chemoattractants CXCL9/MIG, CXCL10/IP-10 and CXCL12/SDF-1 with the use of a multiplex chemokine assay. Chemokine levels except SDF-1 were significantly higher in the 3 disease groups than in normal controls. Considering all patients, mean GRO-α levels were 15-fold higher than IL-8 levels and mean IP-10 levels were 22-fold higher than MIG levels. In patients with the same disease activity, AH levels of GRO-α and IP-10 were significantly higher in patients with BD than in patients with VKH disease and HLA-B27-associated uveitis (p=0.0474; p<0.001, respectively). These data suggest that GRO-α and IP-10 are the predominant CXC chemokines involved in neutrophil and activated T lymphocyte chemoattraction in endogenous uveitis, particularly in BD.

In chronic inflammatory reactions such as rheumatoid arthritis and multiple sclerosis, T cells in the inflamed tissue express the chemokine receptors CXCR3 and CCR5, and the chemokine ligands (CCL) of these receptors are present in the inflammatory lesions. However, the contribution of these chemokines to T cell recruitment to sites of inflammation is unclear. In addition, the relative roles of the chemokines that bind CXCR3 (CXCL9, CXCL10, CXCL11) and CCR5 (CCL3, CCL4, CCL5) in this process are unknown. The in vitro chemotaxis and in vivo migration of antigen-activated T lymphoblasts and unactivated spleen T cells to chemokines were examined. T lymphoblasts migrated in vitro to CXCR3 ligands with a relative potency of CXCL10>> CXCL11>> CXCL9, but these cells demonstrated much less chemotaxis to the CCR5 ligands. In vivo, T lymphocytes were recruited in large numbers with rapid kinetics to skin sites injected with CXCL10 and CCL5 and less to CCL3, CCL4, CXCL9, and CXCL11. The combination of CCL5 with CXCL10 but not the other chemokines markedly increased recruitment. Coinjection of interferon-gamma, tumor necrosis factor alpha, and interleukin-1alpha to up-regulate endothelial cell adhesion molecule expression with CXCL10 or CCL5 induced an additive increase in lymphoblast migration. Thus, CXCR3 ligands are more chemotactic than CCR5 ligands in vitro; however, in vivo, CXCL10 and CCL5 have comparable T cell-recruiting activities to cutaneous sites and are more potent than the other CXCR3 and CCR5 chemokines. Therefore, in vitro chemotaxis induced by these chemokines is not necessarily predictive of their in vivo lymphocyte-recruiting activity.

Interferon-inducible protein-10 (IP-10)/CXCL10, which is a ligand for CXC chemokine receptor 3 (CXCR3), is known to be involved in the pathogenesis of pulmonary sarcoidosis. However, the roles of monokine induced by interferon gamma (Mig)/CXCL9 and interferon-inducible T cell alpha chemoattractant (I-TAC)/CXCL11, which are also CXCR3 ligands, remain unclear. Mig/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11 in both bronchoalveolar lavage fluid (BALF) and serum in patients with pulmonary sarcoidosis were measured by enzyme-linked immunosorbent assay (ELISA). The expression of these chemokines in alveolar macrophages was examined using ELISA, quantitative real-time polymerase chain reaction and immunostaining. In BALF, Mig/CXCL9 and IP-10/CXCL10 were significantly elevated in stage II sarcoidosis as compared with the levels in healthy volunteers. In serum, Mig/CXCL9 and I-TAC/CXCL11 were increased in stage II of the disease. The levels of all CXCR3 ligands in BALF were correlated with the numbers of both total and CD4(+) lymphocytes. Alveolar macrophages were stained positive for all CXCR3 ligands and produced increased amounts of these chemokines. Positive staining of the three chemokines was also observed in the epithelioid and giant cells in the sarcoid lungs. These findings suggest that Mig/CXCL9 and I-TAC/CXCL11 as well as IP-10/CXCL10 play important roles in the accumulation of Th1 lymphocytes in sarcoid lungs.

BACKGROUND

Clinical outcomes following trauma depend on the extent of injury and the host's response to injury, along with medical care. We hypothesized that dynamic networks of systemic inflammation manifest differently as a function of injury severity in human blunt trauma.

METHODS

From a cohort of 472 blunt trauma survivors studied following institutional review board approval, three Injury Severity Score (ISS) subcohorts were derived after matching for age and sex: mild ISS (49 patients [33 males and 16 females, aged 42 ± 1.9 years; ISS 9.5 ± 0.4]); moderate ISS (49 patients [33 males and 16 females, aged 42 ± 1.9; ISS 19.9 ± 0.4]), and severe ISS (49 patients [33 males and 16 females, aged 42 ± 2.5 years; ISS 33 ± 1.1]). Multiple inflammatory mediators were assessed in serial blood samples. Dynamic Bayesian Network inference was utilized to infer causal relationships based on probabilistic measures.

RESULTS

Intensive care unit length of stay, total length of stay, days on mechanical ventilation, Marshall Multiple Organ Dysfunction score, prevalence of prehospital hypotension and nosocomial infection, and admission lactate and base deficit were elevated as a function of ISS. Multiple circulating inflammatory mediators were significantly elevated in severe ISS versus moderate or mild ISS over both the first 24 h and out to 7 days after injury. Dynamic Bayesian Network suggested that interleukin 6 production in severe ISS was affected by monocyte chemotactic protein 1/CCL2, monokine inducible by interferon γ (MIG)/CXCL9, and IP-10/CXCL10; by monocyte chemotactic protein 1/CCL2 and MIG/CXCL9 in moderate ISS; and by MIG/CXCL9 alone in mild ISS over 7 days after injury.

CONCLUSIONS

Injury Severity Score correlates linearly with morbidity, prevalence of infection, and early systemic inflammatory connectivity of chemokines to interleukin 6.

Borrelia burgdorferi, the agent of Lyme disease, promotes proinflammatory changes in the endothelium that lead to the recruitment of leukocytes. The host immune response to infection results in increased levels of IFN-gamma in the serum and lesions of Lyme disease patients that correlate with greater severity of disease. Therefore, the effect of IFN-gamma on the gene expression profile of primary human endothelial cells exposed to B. burgdorferi was determined. B. burgdorferi and IFN-gamma synergistically augmented the expression of 34 genes, 7 of which encode chemokines. Six of these (CCL7, CCL8, CX3CL1, CXCL9, CXCL10, and CXCL11) attract T lymphocytes, and one (CXCL2) is specific for neutrophils. Synergistic production of the attractants for T cells was confirmed at the protein level. IL-1beta, TNF-alpha, and LPS also cooperated with IFN-gamma to induce synergistic production of CXCL10 by the endothelium, indicating that IFN-gamma potentiates inflammation in concert with a variety of mediators. An in vitro model of the blood vessel wall revealed that an increased number of human T lymphocytes traversed the endothelium exposed to B. burgdorferi and IFN-gamma, as compared with unstimulated endothelial monolayers. In contrast, addition of IFN-gamma diminished the migration of neutrophils across the B. burgdorferi-activated endothelium. IFN-gamma thus alters gene expression by endothelia exposed to B. burgdorferi in a manner that promotes recruitment of T cells and suppresses that of neutrophils. This modulation may facilitate the development of chronic inflammatory lesions in Lyme disease.

Despite conventional treatment modalities, gallbladder cancer (GBC) remains a highly lethal malignancy. Prognostic biomarkers and effective adjuvant immunotherapy for GBC are not available. In the recent past, immunotherapeutic approaches targeting tumor associated inflammation have gained importance but the mediators of inflammatory circuit remain unexplored in GBC patients. In the current prospective study, we investigated the role of IL17 producing TCRγδ(+) (Tγδ17), CD4(+) (Th17), CD8(+) (Tc17) and regulatory T cells (Tregs) in pathogenesis of GBC. Analysis by multi-color flow cytometry revealed that compared to healthy individuals (HI), Tγδ17, Th17 and Tc17 cells were increased in peripheral blood mononuclear cells (PBMCs) and tumor infiltrating lymphocytes (TIL) of GBC patients. Tregs were decreased in PBMCs but increased in TILs of GBC patients. The suppressive potential of Tregs from GBC patients and HI were comparable. Serum cytokines profile of GBC patients showed elevated levels of cytokines (IL6, IL23 and IL1β) required for polarization and/or stabilization of IL17 producing cells. We demonstrated that Tγδ17 cells migrate toward tumor bed using CXCL9-CXCR3 axis. IL17 secreted by Tγδ17 induced productions of vascular endothelial growth factor and other angiogenesis related factors in GBC cells. Tγδ17 cells promote vasculogenesis as studied by chick chorioallantoic membrane assay. Survival analysis showed that Tγδ17, Th17 and Treg cells in peripheral blood were associated with poor survival of GBC patients. Our findings suggest that Tγδ17 is a protumorigenic subtype of γδT cells which induces angiogenesis. Tγδ17 may be considered as a predictive biomarker in GBC thus opening avenues for targeted therapies.

In teleost fish two IFN-gamma gene sequences were found for which two phylogenetic clusters can be distinguished. Our previous analysis of expression of these in carp led us to hypothesize that a classical IFN-gamma function is associated with the IFN-gamma2 cluster. We investigated the evolutionary conservation of the IFN-gamma function, inducing classical activation of phagocytes, thus skewing towards a Th1-like profile of immune activation. Recombinant proteins for the carp IFN-gamma sequences of both clusters were made and we studied their effects on expression of proinflammatory mediators. Carp IFN-gamma2, in contrast to carp IFN-gamma1, was powerful in inducing a proinflammatory reaction in phagocytes: a classical synergistic response with lipopolysaccharide was observed for the induction of iNOS expression and NO release, for expression of CXCL9-11-like chemokines and the expression of proinflammatory cytokines IL-1beta, TNFalpha and the IL-12 subunits p35 and p40. In contrast, like in mammals, the CXCL8-like cytokines are LPS but not IFN-gamma sensitive. These results corroborate an evolutionary conserved nature of IFN-gamma function in lower vertebrates including classical activation of phagocytes.

Cysteine cathepsins are primarily lysosomal proteases involved in general protein turnover, but they also have specific proteolytic functions in antigen presentation and bone remodeling. Cathepsins are most stable at acidic pH, although growing evidence indicates that they have physiologically relevant activity also at neutral pH. Post-translational proteolytic processing of mature chemokines is a key, yet underappreciated, level of chemokine regulation. Although the role of selected serine proteases and matrix metalloproteases in chemokine processing has long been known, little has been reported about the role of cysteine cathepsins. Here we evaluated cleavage of CXC ELR (CXCL1, -2, -3, -5, and -8) and non-ELR (CXCL9-12) chemokines by cysteine cathepsins B, K, L, and S at neutral pH by high resolution Tris-Tricine SDS-PAGE and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Whereas cathepsin B cleaved chemokines especially in the C-terminal region, cathepsins K, L, and S cleaved chemokines at the N terminus with glycosaminoglycans modulating cathepsin processing of chemokines. The functional consequences of the cleavages were determined by Ca(2+) mobilization and chemotaxis assays. We show that cysteine cathepsins inactivate and in some cases degrade non-ELR CXC chemokines CXCL9-12. In contrast, cathepsins specifically process ELR CXC chemokines CXCL1, -2, -3, -5, and -8 N-terminally to the ELR motif, thereby generating agonist forms. This study suggests that cysteine cathepsins regulate chemokine activity and thereby leukocyte recruitment during protective or pathological inflammation.

BACKGROUND

The causes of post-Lyme disease symptoms are unclear. Herein, we investigated whether specific immune responses were correlated with such symptoms.

METHODS

The levels of 23 cytokines and chemokines, representative of innate and adaptive immune responses, were assessed in sera from 86 antibiotic-treated European patients with erythema migrans, 45 with post-Lyme symptoms and 41 without symptoms, who were evaluated prior to treatment and 2, 6, and 12 months thereafter.

RESULTS

At study entry, significant differences between groups were observed for the type 1 helper T cell (TH1)-associated chemokines CXCL9 and CXCL10, which were associated with negative Borrelia cultures, and the type 17 helper T cell (TH17)-associated cytokine interleukin 23 (IL-23), which was associated with positive cultures and the development of post-Lyme symptoms (P ≤ .02). Moreover, of the 41 patients with detectable IL-23 levels, 25 (61%) developed post-Lyme symptoms, and all 7 with IL-23 levels ≥ 230 ng/mL had such symptoms. Furthermore, antibody responses to the ECGF autoantigen were more common in patients with post-Lyme symptoms (P = .07) and were correlated directly with IL-23 levels (P = .02). Despite the presence of post-Lyme symptoms, all posttreatment culture results were negative, antiborrelial antibody responses declined, and there were no objective signs of disseminated disease, suggesting that spirochetal eradication had occurred with treatment in all patients.

CONCLUSIONS

High TH1-associated responses correlated with more effective immune-mediated spirochetal killing, whereas high TH17-associated immune responses, often accompanied by autoantibodies, correlated with post-Lyme symptoms, providing a new paradigm for the study of postinfectious symptoms in a subset of patients with Lyme disease.

Recent studies suggest a powerful prognostic value for plasma cytokine levels in primary myelofibrosis (interleukin (IL)-2R, IL-8, IL-12, IL-15 and C-X-C motif chemokine 10 (CXCL10)) and large-cell lymphoma (IL-2R, IL-8, IL-10, IL-12, CXCL9 and CXCL10). To examine the possibility of a similar phenomenon in myelodysplastic syndromes (MDS), we used multiplex enzyme-linked immunosorbent assay to measure 30 plasma cytokines in 78 patients with primary MDS. Compared with normal controls (n = 35), the levels of 19 cytokines were significantly altered. Multivariable analysis identified increased levels of CXCL10 (P<0.01), IL-7 (P = 0.02) and IL-6 (P = 0.07) as predictors of shortened survival; the survival association remained significant when the Cox model was adjusted for the International Prognostic Scoring System, age, transfusion-need or thrombocytopenia. MDS patients with normal plasma levels of CXCL10, IL-7 and IL-6 lived significantly longer (median survival 76 months) than those with elevated levels of at least one of the three cytokines (median survival 25 months) (P<0.01). Increased levels of IL-6 were associated with inferior leukemia-free survival, independent of other prognostic factors (P = 0.01). Comparison of plasma cytokines between MDS (n = 78) and primary myelofibrosis (n = 127) revealed a significantly different pattern of abnormalities. These observations reinforce the concept of distinct and prognostically relevant plasma cytokine signatures in hematological malignancies.

Bone marrow-derived mesenchymal stem cells (MSCs) can localize in injured, inflamed, and cancerous tissues after systemic infusion. However, the dynamic homing profile of MSCs in the peripheral blood is not well characterized. Here, using in vivo flow cytometry to noninvasively monitor the dynamics of fluorescence-labeled cells, we found different clearance kinetics of systemically infused MSCs between healthy and tumor mouse models. The circulation times of MSCs in healthy mice and mice with subcutaneous tumors, orthotopically transplanted liver tumors, or metastatic lung tumors were 30, 24, 18, and 12 hours, respectively, suggesting that MSCs actively home to tumor environments. MSCs infiltrated into hepatocellular carcinoma (HCC) sites and preferentially engrafted to micrometastatic regions both in vivo and in vitro. The expression of epidermal growth factor, CXCL9, CCL25, and matrix metalloproteinases-9 by HCC cells differed between primary tumor sites and metastatic regions. By characterizing the homing profiles of systemically perfused MSCs under physiological and cancerous conditions, these findings increase our understanding of the migration of MSCs from the circulation to tumor sites and constitute a basis for developing MSC-based anti-cancer therapeutic strategies. Stem Cells Translational Medicine 2017;6:1120-1131.

Cryptococcal infections are primarily caused by two related fungal species: Cryptococcus neoformans and Cryptococcus gattii. It is well known that C. neoformans generally affects immunocompromised hosts; however, C. gattii infection can cause diseases in not only immunocompromised hosts but also immunocompetent individuals. While recent studies suggest that C. gattii infection could dampen pulmonary neutrophil recruitment and inflammatory cytokine production in immunocompetent hosts, the impact of C. gattii infection on the development of their adaptive T helper cell immune response has not been addressed. Here, we report that C. neoformans infection with highly virulent and less virulent strains preferentially induced pulmonary Th1 and Th17 immune responses in the host, respectively. However, fewer pulmonary Th1 and Th17 cells could be detected in mice infected with C. gattii strains. Notably, dendritic cells (DC) in mice infected with C. gattii expressed much lower levels of surface MHC-II and Il12 or Il23 transcripts and failed to induce effective Th1 and Th17 differentiation in vitro. Furthermore, the expression levels of Ip10 and Cxcl9 transcripts, encoding Th1-attracting chemokines, were significantly reduced in the lungs of mice infected with the highly virulent C. gattii strain. Thus, our data suggest that C. gattii infection dampens the DC-mediated effective Th1/Th17 immune responses and downregulates the pulmonary chemokine expression, thus resulting in the inability to mount protective immunity in immunocompetent hosts.

BACKGROUND

Increased cytokine and chemokine levels are associated with cardiovascular events in patients with non-ST-elevation acute coronary syndromes (ACS), but the incremental prognostic value of these inflammatory markers is not known. We determined if cytokine and chemokine assessment adds prognostic information to the GRACE Score in patients with ACS.

METHODS

Five cytokines (interleukin (IL)-1beta, IL-6, IL-10, IL-12p70, and tumor necrosis factor (TNF)-alpha soluble receptor I), five chemokines (IL-8, CCL5, CXCL9, CCL2, and CXCL10) and C-reactive protein (CRP) were measured at admission of 87 patients admitted with ACS.

RESULTS

During hospitalization, the incidence of cardiovascular events was 13% (7 deaths, 1 nonfatal acute myocardial infarction, and 3 refractory unstable angina). Individuals who developed events had significantly greater levels of CRP, IL-1beta, IL-12, TNF-alpha, IL-8, CXCL9 and CCL2, compared with those free of events. Thus, these markers were used to build an Inflammatory Score, by the input of one point for each of these variables above the 75th percentile. After adjustment for the GRACE Score, the Inflammatory Score independently predicted events (OR=1.80; 95% CI=1.12-1.88). Incorporation of the Inflammatory Score into the GRACE Score promoted a C-statistics improvement from 0.77 (95% CI=0.58-0.96) to 0.85 (95% CI=0.71-1.0). Net reclassification improvement obtained with GRACE-Inflammatory Score was 13% (P=0.007), indicating a significant reclassification. When only CRP was incorporated into GRACE, the increase on C-statistics was not relevant (from 0.77 to 0.80).

CONCLUSIONS

Cytokines and chemokines measured at admission add prognostic information to the GRACE Score in patients admitted with ACS.

To determine the clinical utility of serum levels of chemokines and cytokines for the evaluation of disease activity in patients with systemic sclerosis (SSc), concentrations of four chemokines (interferon γ-inducible protein-10 [IP-10, CXCL10], monokine induced by interferon γ [MIG/CXCL9], monocyte chemoattractant protein-1 [MCP-1/CCL2], interleukin 8 [IL-8/CXCL8]) and six cytokines (IL-2, IL-4, IL-6, IL-10, tumor necrosis factor [TNF]-α, interferon [IFN]- γ) were measured using cytometric beads array kits in serum samples from 31 Japanese patients with SSc and 20 normal controls. Clinical and laboratory data and serum chemokine and cytokine levels were assessed for each patient at their first visit and each subsequent year for 3 years. Among these chemokines and cytokines, serum levels of IP-10, MIG and MCP-1 were significantly elevated in SSc patients compared with normal controls at their first visit. Serum MCP-1 levels declined year and year, along with improvement for skin sclerosis. The variations of MCP-1, but not IP-10 and MIG, were significantly associated with the variations of skin thickness score and vital capacity during 3 years. These results suggest that MCP-1 is a serological indicator of the activity of skin and lung involvement in patients with SSc. However, a longer-term prospective study in a larger population will be needed to confirm its clinical utility as predictors of outcomes.

IL-1 is a potent pro-inflammatory cytokine that activates intracellular signaling cascades some of which may involve IL-1 receptor associated kinase-1 (IRAK1). Psoriasis is a T cell dependent chronic inflammatory condition of the skin of unknown cause. IL-1 has been implicated in psoriasis pathology, but the mechanism has not been elucidated. Interestingly, expression of IRAK1 is elevated in psoriatic skin. To identify a potential link between IL-1, keratinocytes and T cells in skin inflammation we employed pathway-focused microarrays to evaluate IL-1 dependent gene expression in keratinocytes. Several candidate mRNAs encoding known T cell chemoattractants were identified in primary keratinocytes and the stable keratinocyte cell line HaCaT. CCL5 and CCL20 mRNA and protein levels were confirmed up-regulated by IL-1 in concentration and time-dependent manners. Furthermore IL-1 synergized with IFN-gamma and TNF-alpha. Expression of CXCL9, CXCL10 and CXCL11 mRNAs was also increased in response to IL-1, but protein could only be detected in medium from cells treated with IFN-gamma alone or in combination with IL-1. Over-expression of IRAK1 led to increased constitutive and cytokine induced production of CCL5 and CCL20. Inhibition of IRAK1 activity through RNAi or expression of a dominant negative mutant blocked production of CCL5 and CCL20 but had no effect upon the IL-1 enhancement of IFN-gamma induced CXCL9, CXCL10 and CXCL11 production. In conclusion IL-1 regulates T cell targeting chemokine production in keratinocytes through IRAK1 dependent and independent pathways. These pathways may contribute to acute and chronic skin inflammation.

OBJECTIVE

The chemokine receptor CXCR3 directs migration of T-cells in response to the ligands CXCL9/Mig, CXCL10/IP-10 and CXCL11/I-TAC. Both ligands and receptors are implicated in the pathogenesis of inflammatory disorders, including atherosclerosis and rheumatoid arthritis. Here, we describe the molecular mechanism by which two synthetic small molecule agonists activate CXCR3.

METHODS

As both small molecules are basic, we hypothesized that they formed electrostatic interactions with acidic residues within CXCR3. Nine point mutants of CXCR3 were generated in which an acidic residue was mutated to its amide counterpart. Following transient expression, the ability of the constructs to bind and signal in response to natural and synthetic ligands was examined.

RESULTS

The CXCR3 mutants D112N, D195N and E196Q were efficiently expressed and responsive in chemotaxis assays to CXCL11 but not to CXCL10 or to either of the synthetic agonists, confirmed with radioligand binding assays. Molecular modelling of both CXCL10 and CXCR3 suggests that the small molecule agonists mimic a region of the '30s loop' (residues 30-40 of CXCL10) which interacts with the intrahelical CXCR3 residue D112, leading to receptor activation. D195 and E196 are located in the second extracellular loop and form putative intramolecular salt bridges required for a CXCR3 conformation that recognizes CXCL10. In contrast, CXCL11 recognition by CXCR3 is largely independent of these residues.

CONCLUSIONS

We provide here a molecular basis for the observation that CXCL10 and CXCL11 are allosteric ligands of CXCR3. Such findings may have implications for the design of CXCR3 antagonists.

Herpes simplex virus type 1 (HSV-1) is a human pathogen that may cause severe encephalitis. The development of experimental models of HSV-1 encephalitis is relevant for the comprehension of the immune mechanisms involved in this infection. C57BL/6 mice were inoculated intracranially with 10(4) PFU of neurotropic HSV-1. All animals developed signs of encephalitis and died until day 6 post-infection (pi). Using intravital microscopy, we demonstrated increased leukocyte rolling and adhesion in the brain microvasculature of infected mice at days 1, 3 and 5 pi. The infection was followed by a significant increase in chemokine levels, including CCL2, CCL3, CCL5, CXCL1 and CXCL9. TNF-alpha also showed a significant increase at day 3 pi. Histological analyses demonstrated diffuse meningoencephalitis characterized mainly by mononuclear cell infiltrates. The present model of HSV-1 encephalitis exhibits high mortality in the very first days of infection. Accordingly, there were increased rolling and adhesion of leukocytes along the brain endothelium wall and a high expression of chemokines in the central nervous system. These results corroborate the role of chemokines in leukocyte recruitment following HSV-1 infection in the central nervous system.

BACKGROUND

The survival rate of corneal allografts in high-risk vascularized corneal bed recipients is poor, similar to vascularized solid organ allografts. Although the early induction of selective chemokines in solid organs is required for the optimal recruitment of T cells into rejecting allografts, little is known about the role of these chemokines in high risk corneal allografts.

METHODS

Orthotopic corneal allotransplants were performed in low-risk (nonvascularized) and high-risk (vascularized) C57BL/6 (H-2b) recipients using Balb/c (H-2d) donors. Intragraft production of CXC chemokines was measured by Luminex and enzyme-linked immunosorbent assay on corneal transplant extracts at different times after surgery. Rabbit anti-KC serum was used to test its role in high risk corneal allograft survival.

RESULTS

Early upregulation of CXCL1/KC occurs 3 days after transplantation in high risk allograft only. Moreover, the T-cell chemoattractants, CXCL9/Mig and CXCL10/IP10, are produced late (day 10) after surgery and their production correlates with the recruitment of CD4 T cells into the graft. Furthermore, in vivo neutralization of CXCL1/KC with anti-KC sera results in increased graft survival and decreased recruitment of T cells into high-risk allografts.

CONCLUSIONS

We propose that a high risk vascularized cornea behaves like a vascularized solid organ transplant. The early production of CXCL1/KC is crucial to the induction of T-cell chemoattractants necessary for the recruitment of allospecific CD4 T cells into the graft. In vivo neutralization of CXCL1/KC represents a potential novel therapy that could be used to increase the survival rate of high-risk vascularized corneal allografts.

BACKGROUND

Colorectal cancer is the third most common cancer in the world, a small fraction of which is represented by locally advanced rectal cancer (LARC). If not medically contraindicated, preoperative chemoradiotherapy, represent the standard of care for LARC patients. Unfortunately, patients shows a wide range of response rates in which approximately 20% has a complete pathological response, whereas in 20 to 40% the response is poor or absent.

RESULTS

The following specific gene signature, able to discriminate responders' patients from non-responders, were founded: AKR1C3, CXCL11, CXCL10, IDO1, CXCL9, MMP12 and HLA-DRA. These genes are mainly involved in immune system pathways and interact with drugs traditionally used in the adjuvant treatment of rectal cancer.

CONCLUSIONS

The present study suggests that new ideas for therapy could be found not only limited to studying genes differentially expressed between the two groups of patients but deepening the mechanisms, associated to response, in which they are involved.

METHODS

Gene expression studies performed by: Agostini et al., Rimkus et al. and Kim et al. have been merged through a meta-analysis of the raw data. Gene expression data-sets have been processed using A-MADMAN. Common differentially expressed gene (DEG) were identified through SAM analysis. To further characterize the identified DEG we deeply investigated its biological role using an integrative computational biology approach.

Autoimmune hepatitis lacks an identifiable cause, and its diagnosis requires the exclusion of etiologically defined diseases that resemble it. Insights into its pathogenesis are moving autoimmune hepatitis from an idiopathic to explainable disease, and the goal of this review is to describe the insights that are hastening this transition. Two types of autoimmune hepatitis are justified by serological markers, but they also have distinctive genetic associations (DRB1 and DQB1 genes) and autoantigens. DRB1 alleles are the principal susceptibility factors in white adults, and a six amino acid sequence encoded in the antigen-binding groove of class II molecules of the major histocompatibility complex can influence the selection of autoantigens. Polymorphisms, including variants of SH2B3 and CARD10 genes, may affect immune reactivity and disease severity. The cytochrome mono-oxygenase, CYP2D6, is the autoantigen associated with type 2 autoimmune hepatitis, and it shares homologies with multiple viruses that might promote self-intolerance by molecular mimicry. Chemokines, especially CXCL9 and CXCL10, orchestrate the migration of effector cells to sites of injury and are associated with disease severity. Cells of the innate and adaptive immune responses promote tissue damage, and possible deficiencies in the number and function of regulatory T cells may facilitate the injurious process. Receptor-mediated apoptosis is the principal mechanism of hepatocyte loss, and cell-mediated and antibody-dependent mechanisms of cytotoxicity also contribute. Insights that explain autoimmune hepatitis will allow triggering exogenous antigens to be characterized, risk management to be improved, prognostic indices to be refined, and site-specific therapeutic interventions to emerge.