Esophageal cancer (EC) is one of the most lethal malignancies of the digestive tract and remains to be improved poor prognosis. Two histological subtypes, esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC), are major characteristics of EC. Deep understanding about both subtypes is essential to overcome EC. Here, we focus on chemokines and their receptors as biomarkers and their current applications for the prognosis in EC. We reviewed relevant articles identified using PubMed database for the chemokines and their receptors in EC analyzed by immunohistochemistry. The primary objective is to summarize evidences for them as prognostic biomarkers in EC. A total of twenty-one articles were reviewed after exclusion. Most studies have been done in ESCC, and less in EAC. CXCL12 and its receptor CXCR4 have been shown in both subtypes as biomarkers. CXCR7, CXCL8 and its receptor CXCR2, and CCL21 and its receptor CCR7 have been examined in ESCC. Although it was a small number of reports, CXCL10, CCL4, and CCL5 have been indicated to have anti-tumor effects in ESCC. Chemokines and their receptors have the potential to be the biomarkers in EC. Comparative studies between ESCC and EAC will reveal the similarity and difference in these two subtypes of EC. These studies may indicate whether these molecules play important roles in both subtypes or are unique to one or another.

Background: The aims of this study were to investigate the expression pattern of CDK12 protein in gastric cancer, and to analyze the correlations of CDK12 expression between CD8⁺ cell density and CCL12 expression. Methods: Eighty-six paired tumor and non-tumor samples were collected from patients who underwent radical surgery and had pathological confirmed gastric adenocarcinoma. Immunohistochemistry was used to assess CDK12 expression and CD8⁺ cell density. Expression of CDK12 and CCL21 mRNA was detected by quantitative reverse transcription-polymerase chain reaction. Results: CDK12 expression in gastric tumor tissues was significantly higher than it in paired non-tumor tissues (P<0.001). High expression of CDK12 was identified in 43 cases (50%), and it was significantly correlated with Lauren's classification (diffuse type) and number of metastatic lymph nodes (≥15). High CDK12 protein level indicated a relative poorer overall survival than patients with CKD12 low expression, while it was not identified as an independent prognostic factor. Median number of CD8⁺ cells in tumor tissues was 51 (range: 0-292). Number of CD8⁺ cells was positively correlated with CDK12 expression score in tumor tissues (r=0.243, P=0.024). Positive correlation was also found between CDK12 and CCL21 mRNA expression (r=0.419, P=0.017). Conclusion: High CDK12 expression was detected in gastric cancer which was correlated with malignant phenotypes and worse outcome. Positive correlations of CD8⁺ cell number and CCL21 mRNA expression with CDK12 level were identified.

Background: Inflammatory bowel disease (IBD) is characterized by both acute and chronic phase inflammation of the gastro-intestinal (GI) tract that affect a large and growing number of people worldwide with little to no effective treatments. This is in part due to the lack of understanding of the disease pathogenesis and also the currently poorly described involvement of other systems such as the lymphatics. During DSS induced colitis, mice also develop a severe inflammation of terminal ileum with many features similar to IBD. As well as inflammation within the ileum we have previously demonstrated lymphatic remodeling within the mesentery and mesenteric lymph nodes of DSS-treated mice. The lymphatic remodeling includes lymphangiogenesis, lymphatic vessel dilation and leakiness, as well as cellular infiltration into the surrounding tissue and peripheral draining lymph nodes. Methods: Intestinal inflammation was induced in C57BL/6 mice by administration of 2.5% DSS in drinking water for 7 days. Mice were treated with TLR4 blocker C34 or Polymyxin-B (PMXB) daily from days 3 to 7 of DSS treatment via I.P. injection, and their therapeutic effects on disease activity and lymphatic function were examined. TLR activity and subsequent effect on lymphangiogenesis, lymphadenopathy, and mesenteric lymph node cellular composition were assessed. Results: DSS Mice treated with TLR4 inhibitor, C34, had a significantly improved disease phenotype characterized by reduced ileal and colonic insult. The change correlated with significant reduction in colonic and mesenteric inflammation, resolved mesenteric lymphangiectasia, and CD103⁺ DC migration similar to that of healthy control. PMXB treatment however did not resolve inflammation within the colon or associated mesenteric lymphatic dysfunction but did however prevent lymphadenopathy within the MLN through alteration of CCL21 gradients and CD103⁺ DC migration. Conclusions: TLR4 appears to mediate several changes within the mesenteric lymphatics, more specifically it is shown to have different outcomes whether stimulation occurs through pathogen derived factors such as LPS or tissue derived DAMPs, a novel phenomenon.

OBJECTIVE

Little is known about the molecular details regarding the contribution of different cell types of the salivary gland to the altered gene expression profile seen in Sjögren's syndrome (SS).

METHODS

Using laser microdissection, tissue samples enriched in acini, ducts and inflammatory foci in subjects with and without SS were isolated for RNA-seq analysis. Gene expression profiles were analysed and selected enriched genes were further examined using real time PCR and by immunofluorescence.

RESULTS

RNA-seq analysis of salivary biopsies from subjects with and without SS revealed marked differences in gene expression occurring in the ductal and infiltrating cells compared to acinar cells. Up-regulated genes in the SS ductal cells included C4A complement and the SLC26A9 ion channel. The inflammatory infiltrate showed the most dramatic differences in gene expression and contained up-regulated genes associated with T-cells, natural killer, dendritic and basophils/mast cells. qPCR with total salivary gland mRNA confirmed the differential mRNA expression of several genes (MMP9, FOL1HB, CCL21, CCR7), thereby validating the approach. Additional immunofluorescence studies demonstrated high expression and co-localisation of CCL21 chemokine and CCR7 chemokine receptor within the SS infiltrates.

CONCLUSIONS

Major gene expression changes in the salivary gland of SS were detected in the ductal and inflammatory cells and not in the acinar cells. Two chemokines involved in immune cell trafficking to secondary lymphoid tissue, CCR7 and CCL21, showed markedly increased expression and may contribute to the recruitment of diverse immune cells to the salivary glands, causing inflammation and loss of secretory function.

The characteristic histopathological feature of mycosis fungoides (MF) and adult T-cell leukemia/lymphoma (ATLL) is epidermotropism. To identify the mechanism for epidermotropism of lymphoma cells, total RNAs were obtained from skin biopsies of epidermis and dermis of MF and ATLL patients by means of laser capture microdissection, and used for subsequent complementary DNA (cDNA) microarray experiments. This procedure has made it possible for us to observe and evaluate the regional environment of MF and ATLL. Hierarchical cluster analysis revealed that the cDNAs could be clearly differentiated into MF and ATLL. CCL27 was expressed in the dermis generated from keratinocytes, CCR4/CCR6/CCR7/CCR10/cutaneous lymphocyte-associated antigen (CLA) lymphoma cells in the dermis, and CCL21 in the extracellular matrix (stroma). Lymphotoxin (LT) β and CCL21 expression was significantly higher and that of CCR10 relatively for MF, while CCR4 and CLA expression was relatively higher for ATLL. In the epithelium, keratinocytes expressed CCL20/CCL27, and lymphoma cells CCR4/CCR6/CCR10, while CCR4, CCR6, CCL20 and CCL27 expression was relatively higher for ATLL than MF. The dermis of MF, but not that of ATLL, showed correlation between CCR7 and CCL21. These findings support the suggestion that chemokines and chemokine receptors are involved in the pathogenesis of MF and ATLL, indicate that cutaneous homing seems to be different for MF and ATLL, and point to the possibility that cutaneous T-cell lymphomas originate in regulatory T cells, especially in the case of ATLL.

Gallbladder cancer (GBC) is a rare disease with high mortality. However, no biomarkers for the carcinogenesis, progression, prognosis, and early diagnosis are clinically available. This study investigated the expressions of cystathionine-β-synthase (CBS) and C-C chemokine receptor 7 (CCR7) protein and their clinical and pathologic significances in gallbladder squamous cell/adenosquamous carcinomas (SC/ASC) and adenocarcinomas (AC). CBS and chemokine ligand 21 (CCL21) expression was measured using immunohistochemistry in 69 SC/ASCs and 146 ACs. A significantly high percentage of patients with an age above 45 years, lymph node metastasis, and invasion was observed in the SCs/ASCs compared with ACs (P<0.05). Both AC and SC/ASC patients with positive CBS and CCL21 expression exhibited a high tumor-lymph node-metastasis stage, lymph node metastasis, and invasion compared with patients with negative CBS and CCL21 expression (P<0.05 or P<0.01). SC/ASC patients with positive CBS expression was prone to have a larger tumor size than those with negative expression (P<0.05). Positive CBS and CCL21 expression correlated with poor differentiation and larger tumor size in AC patients. Positive CBS and CCL21 are closely associated with a decreased overall survival in SC/ASC and AC patients (P<0.05 or P<0.01) and were independent factors for a poor-prognosis. Both CBS and CCL21 showed a good overall diagnostic performance for SC/ASC (AUC=0.742 and AUC=0.764, respectively) and AC (AUC=0.734 and AUC=0.718, respectively). In conclusion, positive CBS and CCL21 expression are closely associated with the clinical severity and poor prognosis in GBC, and can be a marker for the diagnosis of AC and SC/ASC type of GBC.

It has been suggested that CXCR3 is important for nociception. Our experiments were conducted to evaluate involvement of CXCR3 and its ligands (CXCL4, CXCL9, CXCL10, CXCL11/CCL21) in neuropathic pain. Our studies give new evidence that intrathecal administration of each CXCR3 ligand induces pain-like behaviour in naive mice that occurs shortly after injection due to its location of neurons, which is confirmed by immunofluorescent staining. Moreover, intrathecal administrations of CXCL9, CXCL10, CCL21 neutralizing antibodies diminished pain-related behaviour. RT-PCR/Western blot analysis unprecedentedly showed spinal elevated levels of CXCR3 after chronic constriction injury of the sciatic nerve in rats in parallel with different time-course changes of its endogenous ligands. Initially, on day 2 we observed spinal increased levels of CXCL10 and CXCL11 indicating that these chemokines have important roles in triggering neuropathy. Then, on day 7, we observed increased levels of CXCL4, CXCL9, CXCL10. Interestingly, changes in CXCL9 level persisted until day 28, suggesting that these chemokines are responsible for long-term, persistent neuropathy. Additionally, in DRG the CXCL4, CXCL9 were elevated. The results obtained from primary glial cultures, suggests that all CXCR3 ligands can be produced in microglia, but also, except for CXCL4, in astrocytes. We provide the first evidence that in neuropathy chronic intrathecal administration of CXCR3 antagonist, (±)-NBI-74330, attenuates hypersensitivity with concomitant occurrence of microglial and some of CXCR3 ligands activation observed in the spinal cord and/or DRG level. This paper underlies the significance of CXCR3 in neuropathic pain and shows therapeutic potential of its blockade for enhancement of morphine analgesia as the major novelty of this work.

Arsenic remains an important environmental hazard that causes several human cancers. Arsenic-induced Bowen's disease (As-BD), a skin carcinoma in situ, is the most common arsenical cancer. While great strides have been made in our understanding of arsenic carcinogenesis, how host immunity contributes to this process remains unknown. Patients with As-BD have an impaired contact hypersensitivity response. Although impaired T cell activation has been well-documented in arsenical cancers, how dendritic cell (DC), the key cell regulating innate immunity, regulates the immune response in arsenical cancers remains unclear. Using myeloid derived DC (MDDC) from patients with As-BD and normal controls as well as bone marrow derived DC (BMDC) from mice fed with or without arsenic, we measured the migration of DC. As-BD patients showed an impaired CCL21-mediated MDDC migration in vitro. Arsenic-fed mice had defective DC migration toward popliteal lymph nodes when injected with allogenic BMDCs via foot pad. Using skin from As-BD and normal controls, we found an increased expression of STAT3, a transcriptional factor contributing to impaired DC activation. Arsenic induced STAT3 activation and the production of VEGF in keratinocytes. The increase in VEGF was blocked by inhibiting STAT3 with RNA interference or pharmaceutically with JSI-124. While VEGF by itself minimally induced the expression of CD86 and MHC-II in MDDC, arsenic induced-MDDC activation was abolished by VEGF pretreatment. We concluded that the STAT3-VEGF axis in keratinocytes inhibits DC migration in the microenvironment of As-BD, indicating that cellular interactions play an important role in regulating the disease course of arsenical cancers.

Rheumatoid arthritis (RA) is a complex and multifactorial autoimmune disorder with the involvement of multiple genetic and environmental factors. Genome-wide association studies (GWAS) have identified more than 50 RA genetic loci in European populations. Given the anticipated overlap of RA-relevant genes and pathways across different ethnic groups, we sought to replicate 58 GWAS-implicated SNPs reported in Europeans in Pakistani subjects. 1,959 unrelated subjects comprising 1,222 RA cases and 737 controls were collected from three rheumatology facilities in Pakistan. Genotyping was performed using iPLEX or TaqMan® methods. A total of 50 SNPs were included in the final association analysis after excluding those that failed assay design/run or postrun QC analysis. Fourteen SNPs (LINC00824/rs1516971, PADI4/rs2240336, CEP57/rs4409785, CTLA4/rs3087243, STAT4/rs13426947, HLA-B/MICA/rs2596565, C5orf30/rs26232, CCL21/rs951005, GATA3/rs2275806, VPS37C/rs595158, HLA-DRB1/rs660895, EOMES/rs3806624, SPRED2/rs934734, and RUNX1/rs9979383) were replicated in our Pakistani sample at false discovery rate (FDR) of <0.20 with nominal p values ranging from 4.73E-06 to 3.48E-02. Our results indicate that several RA susceptibility loci are shared between Pakistani and European populations, supporting the role of common genes/pathways.

In the paracortex of the lymph node (LN), T zone fibroblastic reticular cells (TRCs) orchestrate an immune response by guiding lymphocyte migration both physically, by creating three-dimensional (3D) cell networks, and chemically, by secreting the chemokines CCL19 and CCL21 that direct interactions between CCR7-expressing cells, including mature dendritic cells and naive T cells. TRCs also enwrap matrix-based conduits that transport fluid from the subcapsular sinus to high endothelial venules, and fluid flow through the draining LN rapidly increases upon tissue injury or inflammation. To determine whether fluid flow affects TRC organization or function within a 3D network, we regenerated the 3D LN T zone stromal network by culturing murine TRC clones within a macroporous polyurethane scaffold containing type I collagen and Matrigel and applying slow interstitial flow (1-23 microm/min). We show that the 3D environment and slow interstitial flow are important regulators of TRC morphology, organization, and CCL21 secretion. Without flow, CCL21 expression could not be detected. Furthermore, when flow through the LN was blocked in mice in vivo, CCL21 gene expression was down-regulated within 2 h. These results highlight the importance of lymph flow as a homeostatic regulator of constitutive TRC activity and introduce the concept that increased lymph flow may act as an early inflammatory cue to enhance CCL21 expression by TRCs, thereby ensuring efficient immune cell trafficking, lymph sampling, and immune response induction.

Chemokine receptors play important roles in the immune system and are linked to several human diseases. Targeting chemokine receptors have so far shown very little success owing to, to some extent, the promiscuity of the immune system and the high degree of biased signaling within it. CCR7 and its two endogenous ligands display biased signaling and here we investigate the differences between the two ligands, CCL21 and CCL19, with respect to their biased activation of CCR7. We use bystander bioluminescence resonance energy transfer (BRET) based signaling assays and Transwell migration assays to determine (A) how swapping of domains between the two ligands affect their signaling patterns and (B) how receptor mutagenesis impacts signaling. Using chimeric ligands we find that the chemokine core domains are central for determining signaling outcome as the lack of β-arrestin-2 recruitment displayed by CCL21 is linked to its core domain and not N-terminus. Through a mutagenesis screen, we identify the extracellular domains of CCR7 to be important for both ligands and show that the two chemokines interact differentially with extracellular loop 2 (ECL-2). By using in silico modeling, we propose a link between ECL-2 interaction and CCR7 signal transduction. Our mutagenesis study also suggests a lysine in the top of TM3, K130^3.26, to be important for G protein signaling, but not β-arrestin-2 recruitment. Taken together, the bias in CCR7 between CCL19 and CCL21 relies on the chemokine core domains, where interactions with ECL-2 seem particularly important. Moreover, TM3 selectively regulates G protein signaling as found for other chemokine receptors.

The chemokines CCL21 and CCL19, through binding of their cognate receptor CCR7, orchestrate lymph node homing of dendritic cells and naïve T cells. CCL21 notably differs from CCL19 via an unstructured 32-residue C-terminal domain. Previously described roles for the CCL21 C-terminus include GAG-binding, spatial localization to lymphatic vessels, and autoinhibitory modulation of CCR7-mediated chemotaxis. While truncation of the C-terminal tail induced chemical shift changes in the folded chemokine domain, the structural basis for its influence on CCL21 function remain largely unexplored. CCL21 concentration-dependent NMR chemical shifts revealed weak, non-physiological self-association that mimics truncation of the C-terminal tail. We generated a series of C-terminal truncation variants to dissect the C-terminus influence on CCL21 structure and receptor activation. Using NMR spectroscopy, we found that CCL21 residues 80-90 mediate contacts with the chemokine domain. In cell-based assays for CCR7 and ACKR4 activation, we also found that residues 92-100 reduced CCL21 potency in calcium flux, cAMP inhibition, and -arrestin recruitment. Taken together, these structure-function studies support a model wherein intramolecular interactions with specific residues of the flexible C-terminus stabilize a less active monomer conformation of the CCL21. We speculate that the autoinhibitory intramolecular contacts between the C-terminal tail and chemokine body are disrupted by GAG binding and/or interactions with the CCR7 receptor to ensure optimal functionality.

Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6-/- mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.

The chemokine system has been reported to be utilized and manipulated by tumor cells in order to promote local tumor growth and distant dissemination. The present study aimed to investigate the expression of three chemokine ligand-receptor axes in lung carcinoma tissues. Tumor and healthy normal tissue samples were obtained from 120 lung carcinoma patients following surgical resection. Immunohistochemistry and reverse transcription quantitative polymerase chain reaction were used in order to identify the protein and messenger (m)RNA expression of chemokines, including chemokine (C-X-C motif) ligand (CXCL)12/stromal cell-derived factor (SDF)-1, CXCL8/interleukin (IL)-8, chemokine (C-C motif) ligand (CCL)19 and CCL21, and the corresponding chemokine receptors, chemokine (C-X-C motif) receptor (CXCR)4, CXCR1, CXCR2 and chemokine (C-C motif) receptor (CCR)7, respectively. The results revealed that compared with the normal lung tissues, lung carcinoma tissues expressed significantly higher mRNA levels of CXCL12/SDF-1, CXCR4, CXCL8/IL-8, CXCR2, CCL19 and CCR7 (P<0.01). In four histological subtypes, adenocarcinoma presented dominant expression of CXCR4, CXCR2, CXCL8/IL-8 and CCL19 (P<0.05). In addition, it was demonstrated that tumor staging was inversely correlated with chemokine receptor CCR7 and CXCR2 mRNA expression as well as positively correlated with CXCL12/SDF-1, CXCL8/IL-8 and CCL19 mRNA levels (P<0.05). Lymph node metastasis presented a positive correlation with CXCR4, CXCR2 and CXCL8/IL-8 expression and a negative correlation with CCL19 and CCR7 expression (P<0.05). Furthermore, vascular invasion was more prevalent in patients with higher expression levels of CXCR4, CCR7 or CCL19 (P<0.01). In conclusion, these data suggested that the ligand-receptor interaction of CXCL8-CXCR2, CXCL12-CXCR4 and CCL19-CCR7 may be involved in the tumorigenesis of lung carcinoma. Higher expression levels of chemokines and lower expression of chemokine receptors indicated poor tumor staging. The CXC chemokine receptors, CXCR4 and CXCR2, promoted lymphatic metastasis through the activation of their specific ligands, while CCL19 and its receptor CCR7 had an essential role in hematogenous metastasis of lung carcinoma.

Innate immune responses are known to influence the subsequent development of adaptive immunity. We have previously shown that RSV infection of human airway epithelial cells results in production of the B cell growth factor, BAFF. To better understand how the airway responds to RSV infection by production of this and other factors to support or enhance local B cell responses to infection, we analysed the lung expression of BAFF and B cell homeostatic chemokines CXCL12, CXCL13, CCL19 and CCL21 in a murine model of RSV infection. Following infection with A2 strain RSV, the highest RSV N gene expression was observed at day 4 after challenge with virus. In contrast, two peaks of elevated BAFF expression at days 2 and 7 were observed. CXCL13 was significantly elevated at days 1, 2 and 7. CXCL12, CCL19 and CCL21 were expressed within lung tissue from control and RSV challenged animals but no significant difference in expression was found. Immunofluorescence showed BAFF to be present throughout the tissue however CXCL13 expression was localized to cell rich areas probably constituting lymphoid aggregates. Our results define the kinetics of B cell chemoattractant and growth factor expression during RSV infection and indicate an important role for these cytokines in the airway response to RSV infection.

Ligustrazine which is isolated from Chinese herb ligusticum chuanxiong hort, has been widely used in traditional Chinese medicine (TCM) for asthma treatment. In this study, we aim to observe the effect of ligustrazine on inflammation and the associated chemokines and receptors in ovalbumin (OVA)-induced mouse asthma model. Our data demonstrates that ligustrazine suppresses airway hyperresponsiveness to methacholine and lung inflammation in OVA-induced mouse asthma model. Ligustrazine also induces inhibition of inflammatory cells including neutrophils, lymphocytes and eosinophils. In addition, ligustrazine significantly reduces IL-4, IL-5, IL-17A, CCL3, CCL19 and CCL21 level in BALF of asthma mice. Furthermore, ligustrazine induces down-regulation of CCL19 receptor CCR7, STAT3 and p38 MAPK protein expression. Collectively, these results suggest that ligustrazine is effective in attenuation of allergic airway inflammatory changes and related chemokines and receptors in OVA-induced asthma model, and this action might be associated with inhibition of STAT3 and p38 MAPK pathway, which indicates that ligustrazine may be used as a potential therapeutic method to treat asthma.

BACKGROUND

Epidermolysis bullosa (EB) is a congenital, refractory skin disease and there are no fundamental treatments. Recently, allogenic cell therapies are beginning to be applied as potential treatments, that are based on the concept that the allogenic cells can migrate into the skin and reconstitute the skin components. Although the mechanisms of cell migration into skin are not fully understood, chemokines are regarded as key factors in recruiting bone marrow-derived cells.

OBJECTIVE

Our study aims to elucidate the expression of chemokines in the EB patients.

METHODS

We determined the expression of wound-healing related chemokines in the sera, keratinocytes, and skin tissues of EB patients and compared them to those of healthy volunteers by enzyme-linked immunosorbent assays, quantitative reverse transcription PCR, and immunofluorescence staining.

RESULTS

The serum levels of CXCL12 and HMGB1 were found to be significantly elevated in the EB patients. Conversely, the serum levels of CCL21 were found to be lower in the EB patients than in healthy controls. In addition, the serum levels of CXCL12 tended to increase and the serum levels of CCL27 tended to decrease with an increase in the affected body surface areas. To detect the origin of the circulating chemokines, we performed immunofluorescence staining. CCL21, CCL27, HMGB1 and CXCL12 were stained more broadly in the EB patient tissues than those in the control tissues.

CONCLUSIONS

These results suggest that fluctuations in chemokine levels may contribute in a coordinated way to the wound-healing process and lend clues toward efficient cell therapies for EB.

Mechanisms controlling leukocyte adhesion, propulsion and directional migration have not been fully integrated. In this issue of Immunity, Schumann et al. (2010) propose that DCs swarm to T cell zones using immobilized CCL21 for adhesive random migration and soluble CCL21 for steering.

Background: Tertiary lymphoid organs (TLOs) occur after multiple chronic kidney injuries. interleukin-17A (IL-17A) has been reported to associate with the development of TLOs in inflammatory diseases. However, regulation of the renal TLOs and its clinical significance to the pathogenesis of chronic kidney injury are unknown. Methods: To evaluate the clinical significance and regulation of renal TLOs, we analyzed the progression of patients with kidney damage based on the existence and absence of TLOs in a larger multicenter cohort. We also blocked the recruitment of lymphocyte cells into the kidney by FTY720 (fingolimod) in vivo. Besides, we used aged IL-17A genetic knocked out mice and IL-17A-neutralizing antibody to explore the role of IL-17A in renal TLOs formation. Results: We demonstrated that renal TLOs of IgA nephropathy patients were associated with disease severity and were independent risk factors for renal progression after adjustment for age, sex, mean arterial pressure, proteinuria and, baseline eGFR and MEST-C score, especially in the early stage. Plasma levels of TLO-related chemokines CXCL13, CCL19, and CCL21 were higher in patients with renal TLOs. Inhibiting the formation of renal TLOs by FTY720 could reduce the intrarenal inflammation and fibrosis, and early intervention was found to be more effective. IL-17A was increased in renal TLOs models, and genetic depletion of IL-17A or treatment with anti-IL-17A antibody resulted in a marked reduction of the TLOs formation as well as alleviation of renal inflammation and fibrosis. Conclusion: These results indicate that TLOs are associated with the progression of kidney damage and regulated by IL-17A and may be effective targets for the treatment of kidney damage.

Keywords: IL-17A; Tertiary lymphoid organs; inflammation; kidney damage; progression.

Dyslipidemia is a central component of atherosclerosis and metabolic syndrome linked to chronic inflammation and immune dysfunction. Previously, we showed that hypercholesterolemic apolipoprotein E knock out (apoE^-/-) mice exhibit systemic effects including skin inflammation and hypertrophic lymph nodes (LNs). However, the mechanisms accounting for LN hypertrophy in these mice remain unknown. Here, we show that hypercholesterolemia led to the accumulation of lymphocytes in LNs. We excluded that the increased number of lymphocytes in expanded LNs resulted from increased lymphocyte proliferation or entry into those LNs. Instead, we demonstrated that the egress of lymphocytes from the enlarged LN of apoE^-/- mice was markedly decreased. Impairment in efferent lymphatic emigration of lymphocytes from LNs resulted from an aberrant expansion of cortical and medullary sinuses that became hyperplastic. Moreover, CCL21 was more abundant on these enlarged sinuses whereas lymph levels of sphingosine 1 phosphate (S1P) were decreased in apoE^-/- mice. Normal LN size, lymphatic density and S1P levels were restored by reversing hypercholesterolemia. Thus, systemic changes in cholesterol can sequester lymphocytes in tissue draining LNs through the extensive remodeling of lymphatic sinuses and alteration of the balance between retention/egress signals leading to LN hypertrophy which subsequently may contribute to poor immunity. This study further illustrates the role of lymphatic vessels in immunity through the regulation of immune cell trafficking.

The ability to engineer immune function has transformed modern medicine, highlighted by the success of vaccinations and recent efforts in cancer immunotherapy. Further directions in programming the immune system focus on the design of immunomodulatory biomaterials that can recruit, engage with, and program immune cells locally in vivo. Here, we synthesized shear-thinning and self-healing polymer-nanoparticle (PNP) hydrogels as a tunable and injectable biomaterial platform for local dendritic cell (DC) recruitment. PNP gels were formed from two populations of poly(ethylene glycol)-block-polylactide (PEG-b-PLA) NPs with the same diameter but different PEG brush length (2 or 5 kDa). PEG-b-PLA NPs with the longer PEG brush exhibited improved gel formation following self-assembly and faster recovery after shear-thinning. In all cases, model protein therapeutics were released via Fickian diffusion in vitro, and minor differences in the release rate between the gel formulations were observed. PNP hydrogels were loaded with the DC cytokine CCL21 and injected subcutaneously in a murine model. CCL21-loaded PNP hydrogels recruited DCs preferentially to the site of injection in vivo relative to non-CCL21-loaded hydrogels. Thus, PNP hydrogels comprise a simple and tunable platform biomaterial for in vivo immunomodulation following minimally invasive subcutaneous injection.