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The pathophysiology of post-traumatic arthritis (PTOA) is not fully understood. This study used non-invasive repetitive mechanical loading (ML) mouse models to study biochemical, biomechanical, and pain-related behavioral changes induced in mice. Mouse models reflected the effects of the early stages of PTOA in humans. For the PTOA model, cyclic comprehensive loading (9N) was applied to each mouse’s left knee joint. ML-induced biochemical and molecular changes were analyzed after loading completion. Cartilage samples were examined using gene expression analysis. Tissue sections were used in subsequent OA severity scoring. Biomechanical features and pain-related behavior were studied after 24 h and three weeks post-ML sessions to examine the development of PTOA. The loaded left knee joint showed a greater ROS/RNS signal than the right knee, which was not loaded. There was a significant increase in cartilage damage and MMP activity in the mechanically loaded joints relative to non-loaded control knee joints. Similarly, we found a difference in the viscoelastic tangent, which highlights significant changes in mechanical properties. Biochemical analyses revealed significant increases in total NO, caspase-3 activity, H2O2, and PGE2 levels. Gene expression analysis highlighted increased catabolism (MMP-13, IL-1β, TNF-α) with a concomitant decrease in anabolism (ACAN, COL2A1). Histopathology scores clearly indicated increases in OA progression and synovitis. The gait pattern was significantly altered, suggesting signs of joint damage. This study showed that biomechanical, biochemical, and behavioral characteristics of the murine PTOA groups are significantly different from the control group. These results confirm that the current mouse model can be considered for translational PTOA studies.

Citrullination of proteins plays an important role in protein function and it has recently become clear that citrullinated proteins play a role in immune responses. In this study we examined how citrullinated collagen, an extracellular matrix protein, affects T-cell function during the development of autoimmune arthritis. Using an HLA-DR1 transgenic mouse model of rheumatoid arthritis, mice were treated intraperitoneally with either native type I collagen (CI), citrullinated CI (cit-CI), or phosphate buffered saline (PBS) prior to induction of autoimmune arthritis. While the mice given native CI had significantly less severe arthritis than controls administered PBS, mice receiving cit-CI had no decrease in the severity of autoimmune arthritis. Using Jurkat cells expressing the inhibitory receptor leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1), Western blot analysis indicated that while CI and cit-CI bound to LAIR-1 with similar affinity, only CI induced phosphorylation of the LAIR ITIM tyrosines; cit-CI was ineffective. These data suggest that cit-CI acts as an antagonist of LAIR-1 signaling, and that the severity of autoimmune arthritis can effectively be altered by targeting T cells with citrullinated collagen.

The collagen-induced arthritis mouse model is a widely studied autoimmune model of rheumatoid arthritis. In this model, autoimmune arthritis is induced by immunization of genetically susceptible strains of mice with type II collagen emulsified in complete Freund's adjuvant. This article describes the steps necessary for the acquisition, handling, and preparation of CII, in addition to the selection of mouse strains, proper immunization technique, and methods for evaluation of the incidence and severity of the autoimmune arthritis. In this model, the first signs of arthritis appear approximately 21 to 28 days after immunization. The protocols in this article should provide the investigator with all the necessary information required to reproducibly induce a high incidence of CIA in genetically susceptible strains of mice, and to critically evaluate the pathology of the disease. Published 2021. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol: Induction of collagen-induced arthritis Support Protocol 1: Purification of type II collagen Support Protocol 2: Purification of type II collagen α1(II) chains Support Protocol 3: Assessment of arthritis incidence and severity Support Protocol 4: Measurement of CII specific antibody by indirect ELISA Support Protocol 5: Coupling CII to magnetic beads Support Protocol 6: Measuring CII-specific antibody by magnetic-bead based ELISA Support Protocol 7: Measurement of T cell responses to CII in CIA.

Hepatocellular carcinoma (HCC) is a malignancy with one of the worst prognoses. Long noncoding RNA (lncRNA) are emerging as an important regulator of gene expression and function, leading to the development of cancer. The aim of this study was to determine the relationship between lncRNA and HCC and to further guide clinical therapy. lncRNA in HCC and adjacent tissues were screened, and the correlation between lncRNA‐PDPK2P expression in liver tissues and the pathological characteristics and severity of HCC was assessed. The effects of PDPK2P on HCC proliferation, apoptosis, metastasis, and invasion were also systematically investigated via CCK‐8 assay, flow cytometry, scratch wound healing, and transwell assay, respectively. The relationship between PDPK2P and PDK1 was verified by RNA pull‐down, rescue experiments and western blot. lncRNA‐PDPK2P was highly expressed in HCC tissues with a distinct positive correlation between PDPK2P and PDK1, and the upregulation was clinically associated with a larger tumor embolus, low differentiation, and poor survival. Mechanistically, lncRNA‐PDPK2P interacted with PDK1 and promoted HCC progression through the PDK1/AKT/caspase 3 signaling pathway. lncRNA‐PDPK2P can promote HCC progression, suggesting it may be a clinically valuable biomarker and serve as a molecular target for the diagnosis, prognosis, and therapy of hepatocellular carcinoma.

The ability to use large doses of vitamin D3 (D3) to chronically treat autoimmune diseases such as rheumatoid arthritis (RA) is prohibitive due to its calcemic effect which can damage vital organs. Cytochrome P450scc (CYP11A1) is able to convert D3 into the noncalcemic analog 20S-hydroxyvitamin D3 [20S(OH)D3]. We demonstrate that 20S(OH)D3 markedly suppresses clinical signs of arthritis and joint damage in a mouse model of RA. Furthermore, treatment with 20S(OH)D3 reduces lymphocyte subsets such as CD4+ T cells and CD19+ B cells leading to a significant reduction in inflammatory cytokines. The ratio of T reg cells (CD4+CD25+Foxp3+ T cells) to CD3+CD4+ T cells is increased while there is a decrease in critical complement-fixing anti-CII antibodies. Since pro-inflammatory cytokines and antibodies against type II collagen ordinarily lead to destruction of cartilage and bone, their decline explains why arthritis is attenuated by 20(OH) D3. These results provide a basis for further consideration of 20S(OH)D3 as a potential treatment for RA and other autoimmune disorders.

ObjectiveBiochemical and molecular changes involved in the pathophysiology of post-traumatic arthritis (PTOA) have not been fully understood. This study used non-invasive mouse models to study biochemical, biomechanical and pain-related behavior changes induced in mice following repetitive mechanical knee loading. Mouse models were used to reflect the effects of the early stages of PTOA in humans.MethodsForty-eight twelve week old male mice were obtained for three groups: normal control without mechanical loading, trauma (24 hours after loading), and PTOA (early OA) groups. For the non-invasive PTOA mouse model, cyclic comprehensive loading (9 N) was applied on the left knee joint of each mouse. Biochemical and molecular changes induced by mechanical loading were analyzed after loading was completed. Blood and cartilage were collected and further examined using gene expression analysis. Grading of the tissue sections was completed using the osteoarthritis research society international (OARSI) scale. Biomechanical features of mechanically loaded knee joints were determined after 24 hours (Trauma) and three weeks (PTOA) post-mechanical loading sessions to examine the development of PTOA, respectively.ResultsThe loaded left knee joint showed a greater ROS/RNS signal than the right knee that was not loaded. There was an increase in cartilage damage and MMP activity in the affected knee as the intensity of MabCII680 and MMP750 signal increased in the mechanical loaded joints as compared to unloaded control knee joints. There was also an increase in the difference of viscoelastic energy dissipation ability (tan δ) in PTOA. The OA score increased significantly in mechanically loaded knee joints.ConclusionThis study showed that biomechanical, biochemical, and behavioral characteristics of the murine PTOA groups are significantly different from the control group. These results validate that the current mouse model can be used for translational studies to examine PTOA.

Our previous studies have shown that inoculation of the oral cavity of “humanized” B6.DR1/4 mice with the periodontal pathogen Porphyromonas gingivalis results in an increase in the percentage of circulating Th17 cells, loss of bone and an exacerbation of experimental autoimmune arthritis. The aim of this study was to assess the role played by the human HLA-DRβ molecule containing the shared epitope supplied as a transgene to I-A˚ (murine class II null) C57BL/6 (B6) mice in driving these findings. We compared various immune response parameters as well as alveolar and peri-articular bone loss between humanized B6.DR1 (or B6.DR4) mice and their WT (B6) counterparts. We found that the presence of the shared epitope in the context of inoculation with P. gingivalis enhanced the percentage of Th17 cells generated, dramatically enhanced bone loss and importantly allowed for the generation of CCP2⁺ ACPAs that are not found in C57BL/6 or DBA/1 arthritic mouse serum. Due to the exceedingly complex nature of environmental factors impacting on genetic elements, it has been difficult to unravel mechanisms that drive autoimmune arthritis in susceptible individuals. The findings in this study may provide one small piece of this puzzle that can help us to better understand part of this complexity.

The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly PGE2, are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal PGE synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell (Treg) development. During a type II collagen–CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Ag-experienced mPGES1−/− CD4+ cells showed impaired IL-17A, IFN-γ, and IL-6 production when rechallenged ex vivo with their cognate Ag compared with their wild-type counterparts. Additionally, production of PGE2 by cocultured APCs synergized with that of Ag-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFN-γ responses. However, in contrast with CD4+ cells that were Ag primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFN-γ production under Th17 polarization of naive T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an Ag-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFN-γ responses.

The Cre-LoxP conditional knockout strategy has been used extensively to study gene function in a specific cell-type. In this study, the authors tried to engineer mice in which the Dbc1 gene is conditionally knocked out in Treg cells. Unexpectedly, the conditional Dbc1 allele was completely deleted with a low frequency in some Foxp3YFP-Cre mice harboring floxed Dbc1 allele under specific settings. It was found that the germline recombination of floxed Dbc1 allele, which caused Dbc1 knock out mice, occurred in the male Foxp3YFP-Cre mice harboring floxed Dbc1 allele. Even though the authors documented that Foxp3 is expressed in the testis, the germline recombination was not caused by the germline expression of Cre, which was driven by the Foxp3 promoter. The germline recombination may be caused by the unspecific expression of Cre recombinase in the fetus, in which the floxed Dbc1 allele of some stem cells with development potential to germ cells may be recombined. Additionally, this study found that the floxed Dbc1 allele was recombined in non-T cells of some Foxp3Cre Dbc1fl mice, which need to be characterized. Our results also suggest that using male mice with a low frequency of recombined gene allele can reduce the risk of having full knock out mice.

Multiple observations implicate T-cell dysregulation as a central event in the pathogenesis of rheumatoid arthritis. Here, we investigated mechanisms for suppressing T-cell activation via the inhibitory receptor leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1). To determine how LAIR-1 affects T-cell receptor (TCR) signaling, we compared 1) T cells from LAIR-1–sufficient and –deficient mice, 2) Jurkat cells expressing either LAIR-1 mutants or C-terminal Src kinase (CSK) mutants, and 3) T cells from mice that contain a CSK transgene susceptible to chemical inhibition. Our results indicated that LAIR-1 engagement by collagen or by complement C1q (C1Q, which contains a collagen-like domain) inhibits TCR signaling by decreasing the phosphorylation of key components in the canonical T-cell signaling pathway, including LCK proto-oncogene SRC family tyrosine kinase (LCK), LYN proto-oncogene SRC family tyrosine kinase (LYN), ζ chain of T-cell receptor–associated protein kinase 70 (ZAP-70), and three mitogen-activated protein kinases (extracellular signal–regulated kinase, c-Jun N-terminal kinase 1/2, and p38). The intracellular region of LAIR-1 contains two immunoreceptor tyrosine-based inhibition motifs that are both phosphorylated by LAIR-1 activation, and immunoprecipitation experiments revealed that Tyr-251 in LAIR-1 binds CSK. Using CRISPR/Cas9-mediated genome editing, we demonstrate that CSK is essential for the LAIR-1–induced inhibition of the human TCR signal transduction. T cells from mice that expressed a PP1 analog–sensitive form of CSK (CskAS) corroborated these findings, and we also found that Tyr-251 is critical for LAIR-1's inhibitory function. We propose that LAIR-1 activation may be a strategy for controlling inflammation and may offer a potential therapeutic approach for managing autoimmune diseases.

Toll-like receptor (TLR) signaling can contribute to the pathogenesis of arthritis. Disruption of TLR signaling at early stages of arthritis might thereby provide an opportunity to halt the disease progression and ameliorate outcomes. We previously found that Gö6976 inhibits TLR-mediated cytokine production in human and mouse macrophages by inhibiting TLR-dependent activation of protein kinase D1 (PKD1), and that PKD1 is essential for proinflammatory responses mediated by MyD88-dependent TLRs. In this study, we investigated whether PKD1 contributes to TLR-mediated proinflammatory responses in human synovial cells, and whether Gö6976 treatment can suppress the development and progression of type II collagen (CII)-induced arthritis (CIA) in mouse. We found that TLR/IL-1R ligands induced activation of PKD1 in human fibroblast-like synoviocytes (HFLS). TLR/IL-1R-induced expression of cytokines/chemokines was substantially inhibited in Gö6976-treated HFLS and PKD1-knockdown HFLS. In addition, serum levels of anti-CII IgG antibodies, and the incidence and severity of arthritis after CII immunization were significantly reduced in mice treated daily with Gö6976. Synergistic effects of T-cell receptor and TLR, as well as TLR alone, on spleen cell proliferation and cytokine production were significantly inhibited in the presence of Gö6976. Our results suggest a possibility that ameliorating effects of Gö6976 on CIA may be due to its ability to inhibit TLR/IL-1R-activated PKD1, which might play an important role in proinflammatory responses in arthritis, and that PKD1 could be a therapeutic target for inflammatory arthritis.

Burkitt’s lymphoma (BURK), diffuse large B-cell lymphoma (DLBCL) and mantle cell lymphoma (MCL) are three main types of B-cell lymphomas. This study aimed to compare the differences of affected biological functions and pathways, as well as to explore the possible regulatory mechanisms and the potential therapeutic targets in BURK, DLBCL and MCL. We performed an integrated analysis of 10 lymphoma datasets including 352 BURK patients, 880 DLBCL patients, 216 MCL patients, and 33 controls. Our results showed that signaling pathways, amino acid metabolism and several lipid metabolism pathways varies considerably among these three types of lymphoma. Furthermore, we identified several key transcription factors (TFs) and their target genes that may promote these diseases by influencing multiple carcinogenic pathways. Among these TFs, we reported first that E2F8 displayed the most significant effects in BURK and MCL. Our results demonstrate that over-expression of E2F8 activates target genes that may promote cell cycle, mitosis, immune and other cancer related functions in BURK and MCL. Therefore, we suggest that E2F8 could be used as a biomarker and potential therapeutic target for BURK and MCL. These findings would be helpful in the study of pathogenesis, and drug discovery and also in the prognosis of B cell lymphomas.

Cytochrome P450 (CYP) 1B1 is implicated in vascular smooth muscle cell migration, proliferation, and hypertension. We assessed the contribution of CYP1B1 to angiotensin (Ang) II–induced abdominal aortic aneurysm (AAA). Male Apoe −/− / Cyp1b1 +/+ and Apoe −/− / Cyp1b1 −/− mice were infused with Ang II or its vehicle for 4 weeks; another group of Apoe −/− / Cyp1b1 +/+ mice was coadministered the CYP1B1 inhibitor 2,3′,4,5′-tetramethoxystilbene (TMS) every third day for 4 weeks. On day 28 of Ang II infusion, AAAs were analyzed by ultrasound and ex vivo by Vernier calipers, mice were euthanized, and tissues were harvested. Ang II produced AAAs in Apoe −/− / Cyp1b1 +/+ mice; mice treated with TMS or Apoe −/− / Cyp1b1 −/− mice had reduced AAAs. Ang II enhanced infiltration of macrophages, T cells, and platelets and increased platelet-derived growth factor D, Pdgfrb , Itga2 , and matrix metalloproteinases 2 and 9 expression in aortic lesions; these changes were inhibited in mice treated with TMS and in Apoe −/− / Cyp1b1 −/− mice. Oxidative stress resulted in cyclooxygenase-2 expression in aortic lesions. These effects were minimized in Apoe −/− / Cyp1b1 +/+ mice treated with TMS and in Apoe −/− / Cyp1b1 −/− mice and by concurrent treatment with the superoxide scavenger 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl. CYP1B1 contributed to the development of Ang II–induced AAA and associated pathogenic events in mice, likely by enhancing oxidative stress and associated signaling events. Thus, CYP1B1 may serve as a target for therapeutic agents for AAA in males.

Diagnosis of cartilage damage in early stages of arthritis is vital to impede the progression of disease. In this regard, considerable progress has been made in near-infrared fluorescence (NIRF) optical imaging technique. Arthritis can develop due to various mechanisms but one of the main contributors is the production of matrix metalloproteinases (MMPs), enzymes that can degrade components of the extracellular matrix. Especially, MMP-1 and MMP-13 have main roles in rheumatoid arthritis and osteoarthritis because they enhance collagen degradation in the process of arthritis. We present here a novel NIRF imaging strategy that can be used to determine the activity of MMPs and cartilage damage simultaneously by detection of exposed type II collagen in cartilage tissue. In this study, retro-orbital injection of mixed fluorescent dyes, MMPSense 750 FAST (MMP750) dye and Alexa Fluor 680 conjugated monoclonal mouse antibody immune-reactive to type II collagen, was administered in the arthritic mice. Both dyes were detected with different intensity according to degree of joint destruction in the animal. Thus, our dual fluorescence imaging method can be used to detect cartilage damage as well as MMP activity simultaneously in early stage arthritis.

The aim of this study was to understand how Syk affects peripheral T cell function. T cells from Syk (−/−) chimeric mice and DR1 Syk(fl/fl) CD4(cre) conditional mice gave strong CD3-induced Th1, Th2, and Th17 cytokine responses. However, an altered peptide ligand (APL) of human CII (256 276) with two substitutions (F263N, E266D), also called A12, elicited only Th2 cytokine responses from Syk (fl/fl) T cells but not Syk (fl/fl−)CD4(cre) T cells. Western blots revealed a marked increase in the phosphorylation of Syk, JNK and p38 upon A12/DR1 activation in WT or Syk (fl/fl) T cells but not in Syk(fl)/(fl)CD4(−cre) cells. We demonstrate that Syk is required for the APL- induction of suppressive cytokines. Chemical Syk inhibitors blocked activation of GATA-3 by peptide A12/DR1. In conclusion, this study provides novel insights into the role that Syk plays in directing T cell activity, and may shape therapeutic approaches for autoimmune diseases.

Regulatory T cells (Treg) play a crucial role in maintaining immune homeostasis since Treg dysfunction in both animals and humans is associated with multi-organ autoimmune and inflammatory disease. While IL-2 is generally considered to promote T-cell proliferation and enhance effector T-cell function, recent studies have demonstrated that treatments that utilize low-dose IL-2 unexpectedly induce immune tolerance and promote Treg development resulting in the suppression of unwanted immune responses and eventually leading to treatment of some autoimmune disorders. In the present review, we discuss the biology of IL-2 and its signaling to help define the key role played by IL-2 in the development and function of Treg cells. We also summarize proof-of-concept clinical trials which have shown that low-dose IL-2 can control autoimmune diseases safely and effectively by specifically expanding and activating Treg. However, future studies will be needed to validate a better and safer dosing strategy for low-dose IL-2 treatments utilizing well-controlled clinical trials. More studies will also be needed to validate the appropriate dose of IL-2/anti-cytokine or IL-2/anti-IL-2 complex in the experimental animal models before moving to the clinic., Immunotherapy: Interleukin-2 therapy promising for autoimmune disease Low doses of the immune signaling molecule interleukin-2 (IL-2) could help suppress unwanted immune responses in patients with autoimmunity. Song Zheng from Penn State Health Milton S. Hershey Medical Center, USA and Sun Yat-sen University, and colleagues reviewed the key role that IL-2 plays in the development and function of regulatory T cells, a type of suppressive immune cell that keeps the rest of the immune system in check. The researchers summarized data from proof-of-concept clinical trials, which showed that, at low doses, IL-2 is well tolerated and induces regulatory T cells to address immune imbalances in patients with lupus, type 1 diabetes and other autoimmune disorders. However, the researchers suggest that IL-2 treatments might need to be combined with other immune-modulating strategies to maximize the therapeutic benefit while minimizing side effects.

Factors that drive T cells to signal through differing pathways remain unclear. We have shown that an altered peptide ligand (A9) activates T cells to utilize an alternate signaling pathway which is dependent upon FcRγ and Syk. However, it remains unknown whether the affinity of peptide binding to MHC drives this selection. To answer this question we developed a panel of peptides designed so that amino acids interacting with the p6 and p9 predicted MHC binding pockets were altered. Analogs were tested for binding to I-A q using a competitive binding assay and selected analogs were administered to arthritic mice. Using the collagen-induced arthritis (CIA) model, arthritis severity was correlated with T cell cytokine production and molecular T cell signaling responses. We establish that reduced affinity of interaction with the MHC correlates with T cell signaling through the alternative pathway, leading ultimately to secretion of suppressive cytokines and attenuation of arthritis.

Antibody-mediated rejection (AMR) has emerged as the major cause of renal allograft dysfunction, and more effective strategies need to be explored for improving transplant outcomes. Regulatory T cells (Tregs), consisting of at least natural and induced Treg subsets, suppress effector responses at multiple levels and play a key role in transplantation tolerance. In this study, we investigated the effect of induced Tregs (iTregs) on preventing antibody-mediated renal injury and rejection in a mouse model. We observed that infusion of iTregs markedly attenuated histological graft injury and rejection and significantly improved renal allograft survival. iTregs exhibited a comprehensive ability to regulate immunological disorders in AMR. Firstly, iTreg treatment decreased the levels of circulating anti-donor antibody and the antibody deposition within allografts. Secondly, iTregs significantly reduced cell infiltration including CD4⁺ T cells (including Th1, Th17, Tfh), CD8⁺IFN-γ⁺cells, natural killer cells, B cells and plasma cells which are involved in the process of AMR. Our results also proved a predominance of M1 macrophage to M2 phenotype infiltration in grafts with acute AMR and M1 macrophage could be declined by iTreg treatment. Collectively, our data demonstrate, for the first time, that TGF-β-induced regulatory T cells can attenuate antibody-mediated acute renal allograft injury through multiple immunological modulating pathways. Safety proven by multiple human studies, use of iTregs in prevention of AMR in clinical practice could be expected.

The use of spinal fusion surgery as a treatment for degenerative spinal conditions and chronic back pain is increasing. However, this technique requires use of a bone grafting material to fuse the vertebrae, traditionally autologous bone, which consists of an optimal combination of osteogenic cell precursors, extracellular matrix proteins and mineral components. To date, this remains the ‘gold standard’ material but its supply is limited and is associated with a number of clinical and ethical difficulties; consequently, various combinations of cells with biological scaffold materials have been tested but have failed to achieve fusion rates even comparable to autologous bone. We successfully fabricated a novel collagen-based scaffold using self-organising atelocollagen combined with nano-hydroxyapatite and chondroitin sulphate, cross-linked by microbial transglutaminase. The scaffold was characterised using a range of imaging, chemical composition and thermal analysis techniques. It was found to exhibit appropriate stiffness and suitable pore size for the adhesion, growth and differentiation of MSCs. The low toxicity makes it suitable for clinical application, and its slow degradation profile would enable the scaffold to promote bone growth over an extended period. This material therefore shows promise for clinical use in spinal fusion and other procedures requiring the use of bone grafts.

An understanding of the development and function of T helper (Th) 1, Th17 and regulatory T (Treg) cells is critical toward revealing pro-inflammatory immune responses, especially in autoimmune diseases. However, there is no standard protocol to monitor the development of these cells over time in vivo. This protocol details a method to generate Th1, Th17, and Treg cells in a T cell-induced colitis model in vivo and monitor their dynamic changes, which can be used for further investigations in their development from naive CD4+ T cells in specific gene knockouts and background strains. This protocol starts with the isolation of naive CD4+CD45RBhigh T cells from C57BL/6 IL-17Agfp mice followed by their i.p. injection into recombinase, activating gene-1-deficient (Rag1−/−) mice. At the indicated time points, mice are killed, and mesenteric lymphocytes and murine lamina propria mononuclear cells are isolated and subjected to flow cytometric analysis to assess the development of Th1, Th17, and Treg cells and Th2, Th9, Th22, and follicular T helper (Tfh) cells. This protocol can be completed within 8 weeks.

Several observations implicate a critical role for T cell dysregulation as a central problem in rheumatoid arthritis. We investigated a mechanism for suppressing T cell activation by stimulating a natural inhibitory receptor called leukocyte-associated Ig-like receptor-1 (LAIR-1). The collagen-induced arthritis (CIA) model and DR-1 transgenic mice were used to study the importance of LAIR-1 in autoimmune arthritis. Splenocytes from wild-type or LAIR-1−/− mice were stimulated with soluble anti-CD3 Ab in the presence or absence of α1(II) and supernatants were collected for cytokine analysis. B6.DR1 mice were immunized with type II collagen/CFA to induce arthritis and were treated with either the stimulatory mAb to LAIR-1 or a hamster IgG control. Finally, B6.DR1/LAIR-1−/− and B6.DR1/LAIR-1+/+ mice were challenged for CIA and mean severity scores were recorded thrice weekly. Using splenocytes or purified CD4+ cells that were sufficient in LAIR-1, CD3-induced cytokine secretion was significantly suppressed in the presence of collagen, whereas LAIR-1–deficient splenocytes had no attenuation. Treatment with a stimulatory mAb to LAIR-1 also significantly attenuated CIA in the LAIR+/+ mice. When B6.DR1/LAIR-1−/− mice were immunized with type II collagen they developed more severe arthritis and had a greater percentage of affected limbs than the wild-type mice. These data demonstrate that collagen can suppress the T cell cytokine response through the action of LAIR-1. Treatment with stimulating LAIR-1 Abs suppresses CIA whereas B6.DR1/LAIR-1−/− mice develop more severe arthritis than wild-type controls. These data suggest that LAIR-1 may be a potential therapeutic target for suppressing rheumatoid arthritis.

It is known that ATRA promotes the development of TGF-β-induced CD4+Foxp3+ iTregs, which play a vital role in the prevention of autoimmune diseases; however, the role of ATRA in facilitating the differentiation and function of CD8+Foxp3+ iTregs remains elusive. Using a head-to-head comparison, we found that ATRA promoted expression of Foxp3 and development of CD4+ iTregs, but it did not promote Foxp3 expression on CD8+ cells. Using a standard in vitro assay, we demonstrated that CD8+ iTregs induced by TGF-β and ATRA were not superior to CD8+ iTregs induced by TGF-β alone. In cGVHD, in a typical lupus syndrome model where DBA2 spleen cells were transferred to DBA2xC57BL/6 F1 mice, we observed that both CD8+ iTregs induced by TGF-β and ATRA and those induced by TGF-β alone had similar therapeutic effects. ATRA did not boost but, conversely, impaired the differentiation and function of human CD8+ iTregs. CD8+ cells expressed the ATRA receptor RAR and responded to ATRA, similar to CD4+ cells. We have identified the differential role of ATRA in promoting Foxp3+ Tregs in CD4+ and CD8+ cell populations. These results will help to determine a protocol for developing different Treg cell populations and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

TGF-β and Foxp3 expressions are crucial for the induction and functional activity of CD4(+)Foxp3(+) regulatory T (iTreg) cells. Here, we demonstrate that although TGF-β-primed CD8(+) cells display much lower Foxp3 expression, their suppressive capacity is equivalent to that of CD4(+) iTreg cells, and both Foxp3(-) and Foxp3(+) CD8+ subsets have suppressive activities in vitro and in vivo. CD8(+)Foxp3(-) iTreg cells produce little IFN-γ but almost no IL-2, and display a typical anergic phenotype. Among phenotypic markers expressed in CD8(+)Foxp3(-) cells, we identify CD103 expression particularly crucial for the generation and function of this subset. Moreover, IL-10 and TGF-β signals rather than cytotoxicity mediate the suppressive effect of this novel Treg population. Therefore, TGF-β can induce both CD8(+)Foxp3(-) and CD8(+)Foxp3(+) iTreg subsets, which may represent the unique immunoregulatory means to treat autoimmune and inflammatory diseases.

Collagen type-II is the dominant type of collagen in articular cartilage and chondroitin sulfate is one of the main components of cartilage extracellular matrix. Afibrillar and fibrillar type-II atelocollagen scaffolds with and without chondroitin sulfate were prepared using casting and freeze-drying methods. The scaffolds were characterised to highlight the effects of fibrillogenesis and chondroitin sulfate addition on viscosity, pore structure, porosity and mechanical properties. Microstructure analysis showed that fibrillogenesis increased the circularity of pores significantly in collagen-only scaffolds, whereas with it, no significant change was observed in chondroitin sulfate- containing scaffolds. Addition of chondroitin sulfate to afibrillar scaffolds increased the circularity of the pores and the proportion of pores between 50 and 300 lm suitable for chondrocytes growth. Fourier transform infrared spectroscopy explained the bonding between chondroitin sulfate and afibrillar collagen- confirmed with rheology resultswhich increased the compressive modulus 10-fold to 0.28 kPa. No bonding was observed in other scaffolds and consequently no significant changes in compressive modulus were detected. © Springer Science+Business Media New York 2013.

The integration of inflammatory signals is paramount in controlling the intensity and duration of immune responses. Eicosanoids, particularly prostaglandin E2 (PGE2), are critical molecules in the initiation and resolution of inflammation and in the transition from innate to acquired immune responses. Microsomal prostaglandin E synthase 1 (mPGES1) is an integral membrane enzyme whose regulated expression controls PGE2 levels and is highly expressed at sites of inflammation. PGE2 is also associated with modulation of autoimmunity through altering the IL-23/IL-17 axis and regulatory T cell development. During a type II collagen (CII)-CFA immunization response, lack of mPGES1 impaired the numbers of CD4+ regulatory (Treg) and Th17 cells in the draining lymph nodes. Antigen-experienced mPGES1−/− CD4+ cells showed impaired IL-17A, IFNγ, and IL-6 production when re-challenged ex vivo with their cognate antigen compared to their WT counterparts. Additionally, production of PGE2 by co-cultured antigen presenting cells synergized with that of antigen-experienced CD4+ T cells, with mPGES1 competence in the APC compartment enhancing CD4+ IL-17A and IFNγ responses. However, in contrast to CD4+ cells that were antigen-primed in vivo, exogenous PGE2 inhibited proliferation and skewed IL-17A to IFNγ production under Th17 polarization of naïve T cells in vitro. We conclude that mPGES1 is necessary in vivo to mount optimal Treg and Th17 responses during an antigen-driven primary immune response. Furthermore, we uncover a coordination of autocrine and paracrine mPGES1-driven PGE2 production that impacts effector T cell IL-17A and IFNγ responses.

Rheumatoid arthritis is an autoimmune disorder characterized by T cell dysregulation. We have shown that an altered peptide ligand (A9) activates T cells to use an alternate signaling pathway that is dependent on FcRγ and spleen tyrosine kinase, resulting in downregulation of inflammation. In the experiments described in this study, we have attempted to determine the molecular basis of this paradox. Three major Src family kinases found in T cells (Lck, Fyn, and Lyn) were tested for activation following stimulation by A9/I-Aq. Unexpectedly we found they are not required for T cell functions induced by A9/I-Aq, nor are they required for APL stimulation of cytokines. On the other hand, the induction of the second messenger inositol trisphosphate and the mobilization of calcium are clearly triggered by the APL A9/I-Aq stimulation and are required for cytokine production, albeit the cytokines induced are different from those produced after activation of the canonical pathway. DBA/1 mice doubly deficient in IL-4 and IL-10 were used to confirm that these two cytokines are important for the APL-induced attenuation of arthritis. These studies provide a basis for exploring the effectiveness of analog peptides and the inhibitory T cells they induce as therapeutic tools for autoimmune arthritis.

Background Fingolimod (FTY720) is an immunomodulating drug that inhibits sphingosine-1-phosphate binding and blocks T-cell egress from lymph nodes. We analyzed the effect of FTY720 on the autoimmune T- and B-cell response in autoimmune arthritis and studied the mechanisms by which it alters the function of T cells. Methods Human leukocyte antigen (HLA)-DR1 humanized mice were immunized with type II collagen (CII) and treated with FTY720 three times per week for 3 weeks. Arthritis was evaluated and autoimmune T- and B-cell responses were measured using proliferation assays, enzyme-linked immunosorbent assays, HLA-DR tetramers, and flow cytometry. The functional capacity of regulatory T (Treg) cells from FTY720-treated mice was measured using an in vitro suppression assay, and the role of Treg cells in inhibiting arthritis in FTY720-treated mice was evaluated using mice treated with anti-CD25 to deplete Treg cells. Results Treatment with FTY720 delayed the onset of arthritis and significantly reduced disease incidence. FTY720 did not prevent the generation of a CII-specific autoimmune T-cell response in vivo. However, as the treatment continued, these T cells became unresponsive to restimulation with antigen in vitro, and this anergic state was reversed by addition of interleukin 2. Measurements of CD4+CD25+Foxp3+ cells in the lymph nodes revealed that the ratio of Treg to helper T (Th) cells increased twofold in the FTY720-treated mice, and in vitro assays indicated that the regulatory function of these cells was enhanced. That FTY720 stimulation of Treg cells played a major role in arthritis inhibition was demonstrated by a loss of disease inhibition and restitution of the T-cell proliferative function after in vivo depletion of the Treg cells. Conclusions While FTY720 affects the recirculation of lymphocytes, its ability to inhibit the development of autoimmune arthritis involves several mechanisms, including the enhancement of Treg cell function by increasing the Treg/Th ratio and increased regulatory function on a per-cell basis. FTY720 did not inhibit the development of the autoimmune T-cell response, but disease inhibition appeared to be mediated by Treg cell–mediated suppression of the CII-specific T cells. These data suggest that specific targeting of Treg cells with FTY720 may be a novel therapy for autoimmunity. Electronic supplementary material The online version of this article (doi:10.1186/s13075-015-0909-6) contains supplementary material, which is available to authorized users.

Transferred CD4+CD25+FoxP3+ Treg cells can prevent autoimmune disease, but generally fail to ameliorate established disease. This study was undertaken to compare the effects of antigen-specific Treg cells induced with interleukin-2 (IL-2) and transforming growth factor β (TGFβ) ex vivo (induced Treg [iTreg] cells) to the effects of equivalent expanded thymus-derived natural Treg (nTreg) cells on established collagen-induced arthritis (CIA).CIA was induced in DBA/1 mice by immunization with type II collagen (CII), and before or shortly after immunization, mice were treated with iTreg or nTreg cells that were generated or expanded in vitro. Clinical scores were determined. Inflammatory responses were determined by measuring the levels of anti-CII antibody in the serum and examining the histologic features of the mouse joints. The Th1/Th17-mediated autoreactive response was evaluated by determining the cytokine profile of the draining lymph node (LN) cells of the mice by flow cytometry.Following transfer, nTreg cells exhibited decreased FoxP3 and Bcl-2 expression and decreased suppressive activity, and many converted to Th17 cells. In contrast, transferred iTreg cells were more numerous, retained FoxP3 expression and their suppressive activity in the presence of IL-6, and were resistant to Th17 conversion. Notably, 10 days after the transfer of donor iTreg cells, predominance was shifted from Th17 cells to Treg cells in the draining LNs of recipient mice.These findings provide evidence that transferred TGFβ-induced iTreg cells are more stable and functional than nTreg cells in mice with established autoimmunity. Moreover, iTreg cells can have tolerogenic effects even in the presence of ongoing inflammation. The therapeutic potential of human iTreg cells in subjects with chronic, immune-mediated inflammatory diseases should be investigated.

Interplay between Foxp3(+) regulatory T cells (Treg) and dendritic cells (DCs) maintains immunologic tolerance, but the effects of each cell on the other are not well understood. We report that polyclonal CD4(+)Foxp3(+) Treg cells induced ex vivo with transforming growth factor beta (TGFβ) (iTreg) suppress a lupus-like chronic graft-versus-host disease by preventing the expansion of immunogenic DCs and inducing protective DCs that generate additional recipient CD4(+)Foxp3(+) cells. The protective effects of the transferred iTreg cells required both interleukin (IL)-10 and TGFβ, but the tolerogenic effects of the iTreg on DCs, and the immunosuppressive effects of these DCs were exclusively TGFβ-dependent. The iTreg were unable to tolerize Tgfbr2-deficient DCs. These results support the essential role of DCs in 'infectious tolerance' and emphasize the central role of TGFβ in protective iTreg/DC interactions in vivo.

Summary Immune responses to human leucocyte antigen (HLA) and self-antigen collagen V (Col-V) have been proposed in the pathogenesis of chronic rejection (bronchiolitis obliterans syndrome, BOS) following human lung transplantation (LTx). In this study, we defined the role for the shift in immunodominant epitopes of Col-V in inducing T helper phenotype switch leading to immunity to Col-V and BOS. Sera and lavage from BOS+ LTx recipients with antibodies to Col-V were analysed. Two years prior to BOS, patients developed antibodies to both Col-V,α1(V) and α2(V) chains. However, at clinical diagnosis of BOS, antibodies became restricted to α1(V). Further, lung biopsy from BOS(+) patients bound to antibodies to α1(V), indicating that these epitopes are exposed. Fourteen Col-V peptides [pep1–14, pep1–4 specific to α1(V), pep5–8 to α1,2(V) and pep9–14 to α2(V)] which bind to HLA-DR4 and -DR7, demonstrated that prior to BOS, pep 6, 7, 9, 11 and 14 were immunodominant and induced interleukin (IL)-10. However, at BOS, the response switched to pep1, 4 and 5 and induced interferon (IFN)-γ and IL-17 responses, but not IL-10. The T helper (Th) phenotype switch is accompanied by decreased frequency of regulatory T cells (Tregs) in the lavage. LTx recipients with antibodies to α1(V) also demonstrated increased matrix metalloproteinase (MMP) activation with decreased MMP inhibitor, tissue inhibitor of metalloproteinase (TIMP), suggesting that MMP activation may play a role in the exposure of new Col-V antigenic epitopes. We conclude that a shift in immunodominance of self-antigenic determinants of Col-V results in induction of IFN-γ and IL-17 with loss of tolerance leading to autoimmunity to Col-V, which leads to chronic lung allograft rejection.

Among several pharmacological compounds, Phlebotomine saliva contains substances with anti-inflammatory properties. In this article, we demonstrated the therapeutic activity of salivary gland extract (SGE) of Phlebotomus papatasi in an experimental model of arthritis (collagen-induced arthritis [CIA]) and identified the constituents responsible for such activity. Daily administration of SGE, initiated at disease onset, attenuated the severity of CIA, reducing the joint lesion and proinflammatory cytokine release. In vitro incubation of dendritic cells (DCs) with SGE limited specific CD4+ Th17 cell response. We identified adenosine (ADO) and 5′AMP as the major salivary molecules responsible for anti-inflammatory activities. Pharmacologic inhibition of ADO A2A receptor or enzymatic catabolism of salivary nucleosides reversed the SGE-induced immunosuppressive effect. Importantly, CD73 (ecto-5′-nucleotidase enzyme) is expressed on DC surface during stage of activation, suggesting that ADO is also generated by 5′AMP metabolism. Moreover, both nucleosides mimicked SGE-induced anti-inflammatory activity upon DC function in vitro and attenuated establishment of CIA in vivo. We reveal that ADO and 5′AMP are present in pharmacological amounts in P. papatasi saliva and act preferentially on DC function, consequently reducing Th17 subset activation and suppressing the autoimmune response. Thus, it is plausible that these constituents might be promising therapeutic molecules to target immune inflammatory diseases.

Objective To explore the characteristics of the T cell population that responds to an analog peptide (A9) of type II collagen and regulates autoimmunity, using the collagen-induced arthritis (CIA) model. Methods Analog peptide A9 is a 26–amino acid peptide analogous to the sequence of a segment of type II collagen (CII245–270) but with substitutions at amino acid positions 260 (alanine for isoleucine), 261 (hydroxyproline for alanine), and 263 (asparagine for phenylalanine). We previously showed that A9 profoundly suppressed CIA and immune responses to type II collagen. In order to determine the mechanism of suppression, we used transgenic mice whose T cells express a type II collagen–specific receptor (T cell receptor) and performed passive cell transfer experiments. Results The results demonstrated that suppression of CIA by A9 is dependent on T cells. Using multiparameter flow cytometry, we determined that the cells responsible for suppression were CD4+ and expressed high levels of Fce receptor Iγ chain (FcRγ). To establish the significance of this finding, we obtained mice genetically deficient in FcRγ in order to perform passive transfer experiments. The resulting FcRγ−/− CD4+ T cells, when primed by culture with A9, could not transfer the suppression of arthritis or secrete cytokines in response to A9. Conclusion Taken together, the results of this study suggest that the suppression of arthritis and the Th2 cytokine profile elicited by A9 is dependent on the presence of FcRγ in T cells. These findings are novel and may have therapeutic potential for patients with autoimmune arthritis.

Recent studies have demonstrated that plasticity of naturally occurring CD4+Foxp3+ regulatory T cells (nTregs) may account for their inability to control chronic inflammation in established autoimmune diseases. All-trans retinoic acid (atRA), the active derivative of vitamin A, has been demonstrated to promote Foxp3+ Treg differentiation and suppress Th17 development. In this study, we report a vital role of atRA in sustaining the stability and functionality of nTregs in the presence of IL-6. We found that nTregs treated with atRA were resistant to Th17 and other Th cell conversion and maintained Foxp3 expression and suppressive activity in the presence of IL-6 in vitro. atRA decreased IL-6R expression and signaling by nTregs. Of interest, adoptive transfer of nTregs even from arthritic mice treated with atRA suppressed progression of established collagen-induced arthritis. We suggest that nTregs treated with atRA may represent a novel treatment strategy to control established chronic immune-mediated inflammatory diseases.

Although it is clear that CD4+ T cells play a major role in mediating the pathogenesis of autoimmunity, they often represent only a minor population at the site of inflammation in autoimmune diseases. To investigate the migration and specificity of autoimmune T cells to the inflammatory site, we used the collagen-induced arthritis model to determine the frequency, clonotype, and specificity of T cells that infiltrate arthritic joints. We demonstrate that despite the fact that CD4+ T cells are a minor population of the synovial infiltrate, the CD4+ T cells present are a highly selective subset of the TCR repertoire and, based on CDR3 length polymorphisms, have a limited clonality. Although a similar repertoire of type II collagen (CII)-specific TCR-BV8 and BV14-expressing T cells was found in peripheral lymphoid organs, the clonality of the TCR-BV8 and BV14 T cells that migrate to the arthritic joint generally made up a single CDR3 length. T cell hybridomas produced from these joint-derived cells revealed that many of these infiltrating T cells are CII specific, and the majority recognize mouse CII. These data suggest that despite being a minor population at the site of inflammation, autoantigen-specific T cells are selectively recruited and/or retained in the arthritic joint and may be playing a significant role in the pathogenesis of the autoimmune arthritis. In addition, this model may be very useful for studying the function in situ and the mechanism by which autoimmune T cells are recruited to the site of inflammation.

Hypersensitivity pneumonitis is an interstitial lung disease that results from repeated pulmonary exposure to various organic Ags, including Saccharopolyspora rectivirgula, the causative agent of farmer’s lung disease. Although the contributions of proinflammatory mediators to the disease pathogenesis are relatively well documented, the mechanism(s) involved in the initiation of proinflammatory responses against the causative microorganisms and the contribution of signaling molecules involved in the host immune defense have not been fully elucidated. In the current study, we found that S. rectivirgula induces the activation of protein kinase D (PKD)1 in lung cells in vitro and in vivo. Activation of PKD1 by S. rectivirgula was dependent on MyD88. Inhibition of PKD by pharmacological PKD inhibitor Gö6976 and silencing of PKD1 expression by small interfering RNA revealed that PKD1 is indispensable for S. rectivirgula-mediated activation of MAPKs and NF-κB and the expression of various proinflammatory cytokines and chemokines. In addition, compared with controls, mice pretreated with Gö6976 showed significantly suppressed alveolitis and neutrophil influx in bronchial alveolar lavage fluid and interstitial lung tissue, as well as substantially decreased myeloperoxidase activity in the lung after pulmonary exposure to S. rectivirgula. These results demonstrate that PKD1 is essential for S. rectivirgula-mediated proinflammatory immune responses and neutrophil influx in the lung. Our findings also imply the possibility that PKD1 is one of the critical factors that play a regulatory role in the development of hypersensitivity pneumonitis caused by microbial Ags and that inhibition of PKD1 activation could be an effective way to control microbial Ag-induced hypersensitivity pneumonitis.

Contains fulltext : 88045.pdf (Publisher’s version ) (Open Access) Rheumatoid arthritis is an autoimmune disease with 1% prevalence in the industrialized world. The contributions of the inflammasome components Nlrp3, ASC, and caspase-1 in the pathogenesis of collagen-induced arthritis have not been characterized. Here, we show that ASC(-/-) mice were protected from arthritis, whereas Nlrp3(-/-) and caspase-1(-/-) mice were susceptible to collagen-induced arthritis. Unlike Nlrp3(-/-) and caspase-1(-/-) mice, the production of collagen-specific antibodies was abolished in ASC(-/-) mice. This was due to a significantly reduced antigen-specific activation of lymphocytes by ASC(-/-) dendritic cells. Antigen-induced proliferation of purified ASC(-/-) T cells was restored upon incubation with wild type dendritic cells, but not when cultured with ASC(-/-) dendritic cells. Moreover, direct T cell receptor ligation with CD3 and CD28 antibodies induced a potent proliferation of ASC(-/-) T cells, indicating that ASC is specifically required in dendritic cells for antigen-induced T cell activation. Therefore, ASC fulfills a hitherto unrecognized inflammasome-independent role in dendritic cells that is crucial for T cell priming and the induction of antigen-specific cellular and humoral immunity and the onset of collagen-induced arthritis.

Rat lung allograft rejection is mediated by collagen type V (col(V)) specific T-helper-cell 17 (Th17) cells. Adoptive transfer of these cells is sufficient to induce rejection pathology in isografts, whereas tolerance to col(V) suppresses allograft rejection. Therefore, we tested whether regulatory T cells from tolerant rats could suppress the Th17-mediated rejection in the syngeneic model of lung transplantation.Rats were subjected to syngeneic left lung transplantation, and acute rejection was induced by adoptive transfer of lymph node cells from col(V)-immunized rats. Tolerance was induced by intravenous injection of col(V), and spleen lymphocytes were used for adoptive transfer. CD4+ T cells were depleted using magnetic beads. Lung isografts were analyzed using micro-positron emission tomography imaging and histochemistry. The transvivo delayed type hypersensitivity assay was used to analyze the Th17 response.Adoptive cotransfer of col(V)-specific effector cells with cells from col(V)-tolerized rats suppressed severe vasculitis and bronchiolitis with parenchymal inflammation, and the expression of interleukin (IL)-17 transcripts in mediastinal lymph nodes induced by effector cells alone. Analysis by transvivo delayed type hypersensitivity showed that the reactivity to col(V) was dependent on the presence of tumor necrosis factor-alpha and IL-17 but not interferon-gamma. Depletion of CD4+ T cells from the suppressor cell population abrogated the col(V)-specific protection.Th17-mediated acute rejection after lung transplantation is ameliorated by CD4+ col(V)-specific regulatory T cells. The mechanism for this Th17 suppression is consistent with tolerance induction to col(V). The goal of transplantation treatment, therefore, should target Th17 development and not suppression of T-cell activation by suppressing IL-2.

Rationale: The pathogenesis of primary graft dysfunction (PGD), a serious complication of lung transplantation, is poorly understood. Human studies and rodent models have shown that collagen type V (col[V]), stimulates IL-17–dependent cellular immunity after lung transplantation. Objectives: To determine whether patients with end-stage lung disease develop pretransplant col(V)-specific cellular immunity, and if so, the impact of this response on PGD. Methods: Trans-vivo delayed-type hypersensitivity (TV-DTH) assays were used to evaluate memory T-cell responses to col(V) in 55 patients awaiting lung transplantation. PaO2/FIO2 index data were used to assess PGD. Univariate risk factor analysis was performed to identify variables associated with PGD. Rats immunized with col(V) or irrelevant antigen underwent lung isografting to determine if prior anti-col(V) immunity triggers PGD in the absence of alloreactivity. Measurements and Main Results: We found that 58.8% (10/17) of patients with idiopathic pulmonary fibrosis, and 15.8% (6/38) of patients without idiopathic pulmonary fibrosis tested while on the wait list for a lung transplant were col(V) DTH positive. Col(V) reactivity was CD4+ T-cell and monocyte mediated, and dependent on IL-17, IL-1b, and tumor necrosis factor (TNF)-a .P aO2 /FIO2 indices were impaired significantly 6–72 hours after transplantation in col(V)-reactive versus nonreactive patients. Univariate risk factor analysis identified only preoperative TV-DTH to col(V) and ischemic time as predictors of PGD. Finally, in a rat lung isograft model, col(V) sensitization resulted in significantly lower PaO2 /FIO2 , increased local TNF-a and IL-1b production, and a moderate-to-severe bronchiolitis/ vasculitis when compared with control isografts. Conclusions: The data suggest that activation of innate immunity by col(V)-specific Th-17 memory cells represents a novel pathway to

BACKGROUND: Immunity to type V collagen [col(V)] contributes to lung transplant rejection. Matrix metalloproteases (MMPs), which are induced by transplant-related ischemia-reperfusion injury (IRI), could expose col(V) and regulate local IRI-induced inflammation. METHODS: To test the hypothesis that MMPs induce col(V) exposure and inflammation, Wistar-Kyoto rats were treated with the MMP inhibitor, COL-3, before inducing lung IRI without transplantation, and in parallel studies, Wistar-Kyoto lung donor and recipients were treated with COL-3 pre- and postisograft lung transplantation. RESULTS: Ischemia-reperfusion injury induced growth-related oncogene/CINC-1-dependent neutrophil influx, and up-regulated tumor necrosis factor-alpha. MMP2 and MMP9, induced at 4 and 24 hr after IRI, respectively, were associated with detection of antigenic col(V) in bronchoalveolar lavage and lung interstitium because of MMP-mediated matrix degradation. MMP-inhibitor treatment significantly reduced polymorphonuclear leukocytes, growth-related oncogene/CINC-1, and tumor necrosis factor-alpha; abrogated MMP-9 expression; and resulted in lower levels of antigenic col(V) in bronchoalveolar lavage. In the lung transplant model, inhibiting MMPs in the donor before lung harvest and in the recipient after lung transplantation resulted in improved oxygenation and diminished polymorphonuclear leukocyte influx into the isograft. CONCLUSION: MMP inhibition may be a potential therapy to prevent release of antigenic col(V) and ameliorate IRI in the transplant recipient.

Bronchiolitis obliterans syndrome (BOS), a process of fibro-obliterative occlusion of the small airways in the transplanted lung, is the most common cause of lung transplant failure. We tested the role of cell-mediated immunity to collagen type V [col(V)] in this process. PBMC responses to col(II) and col(V) were monitored prospectively over a 7-year period. PBMCs from lung transplant recipients, but not from healthy controls or col(IV)-reactive Goodpasture's syndrome patients after renal transplant, were frequently col(V) reactive. Col(V)-specific responses were dependent on both CD4+ T cells and monocytes and required both IL-17 and the monokines TNF-alpha and IL-1beta. Strong col(V)-specific responses were associated with substantially increased incidence and severity of BOS. Incidences of acute rejection, HLA-DR mismatched transplants, and induction of HLA-specific antibodies in the transplant recipient were not as strongly associated with a risk of BOS. These data suggest that while alloimmunity initiates lung transplant rejection, de novo autoimmunity mediated by col(V)-specific Th17 cells and monocyte/macrophage accessory cells ultimately causes progressive airway obliteration.

In trying to assess the structural integrity of electrospun type II collagen scaffolds, a modified but new technique for cross-linking collagen has been developed. Carbodiimides have been previously used to cross-link collagen in gels and in lyophilized native tissue specimens but had not been used for electrospun mats until recently. This cross-linking agent, and in particular 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), is of extreme interest, especially for tissue-engineered scaffolds composed specifically of native polymers (e.g., collagen), because it is a zero-length cross-linking agent that has not been shown to cause any cytotoxic reactions. The unique aspect of the cross-linking protocol in this study involves the use of ethanol as the solvent for the cross-linking agent, because the pure collagen electrospun mats immediately disintegrate when placed in an aqueous solution. This study examines 2 concentrations of EDC with and without the addition of N-hydroxysuccinimide to the reaction (which has been shown to result in higher cross-linking yields in aqueous solutions) to test the hypothesis that the use of EDC in a nonaqueous solution will cross-link electrospun type II collagen fibrous matrices in a comparable manner to typical glutaraldehyde fixation protocols. The use of EDC is compared with the cross-linking effects of glutaraldehyde via mechanical testing (uniaxial tensile testing) and biochemical testing (analysis of the percentage of free amino groups). The stress-strain curves of the cross-linked samples demonstrated uniaxial tensile behavior more characteristic of native tissue than do the dry, untreated samples. The heated, 50% glutaraldehyde cross-linking protocol resulted in a mean peak stress of 0.76 MPa, a mean strain at break of 127.30%, and a mean tangential modulus of 0.89 MPa; mean values for the samples treated with the EDC protocols ranged from 0.35 to 0.60 MPa for peak stress, from 111.83 to 159.23% for strain at break, and from 0.57 to 0.92 MPa for tangential modulus. Low and high concentrations (20 mM and 200 mM, respectively) of EDC alone were comparable in extent of cross-linking (29% and 29%, respectively) to the heated 50% glutaraldehyde cross-linking protocol (30% cross-linked).