Thymic regulatory T cells arise via two distinct developmental programs.

The developmental programs that generate a broad repertoire of regulatory T cells (Treg cells) able to respond to both self antigens and non-self antigens remain unclear. Here we found that mature Treg cells were generated through two distinct developmental programs involving CD25+ Treg cell progenitors (CD25+ TregP cells) and Foxp3lo Treg cell progenitors (Foxp3lo TregP cells). CD25+ TregP cells showed higher rates of apoptosis and interacted with thymic self antigens with higher affinity than did Foxp3lo TregP cells, and had a T cell antigen receptor repertoire and transcriptome distinct from that of Foxp3lo TregP cells. The development of both CD25+ TregP cells and Foxp3lo TregP cells was controlled by distinct signaling pathways and enhancers. Transcriptomics and histocytometric data suggested that CD25+ TregP cells and Foxp3lo TregP cells arose by coopting negative-selection programs and positive-selection programs, respectively. Treg cells derived from CD25+ TregP cells, but not those derived from Foxp3lo TregP cells, prevented experimental autoimmune encephalitis. Our findings indicate that Treg cells arise through two distinct developmental programs that are both required for a comprehensive Treg cell repertoire capable of establishing immunotolerance.

Characterization and comparison of innate and adaptive immune responses at vaccine sites in melanoma vaccine clinical trials.

The strength and durability of systemic anti-tumor immune responses induced by cancer vaccines depends on adjuvants to support an immunogenic vaccine site microenvironment (VSME). Adjuvants include water-in-oil emulsions with incomplete Freund's adjuvant (IFA) and combinations of toll-like receptor (TLR) agonists, including a preparation containing TLR4 and TLR9 agonists with QS-21 (AS15). IFA-containing vaccines can promote immune cell accumulation at the VSME, whereas effects of AS15 are largely unexplored. Therefore, we assessed innate and adaptive immune cell accumulation and gene expression at the VSME after vaccination with AS15 and compared to effects with IFA. We hypothesized that AS15 would promote less accumulation of innate and adaptive immune cells at the VSME than IFA vaccines. In two clinical trials, patients with resected high-risk melanoma received either a multipeptide vaccine with IFA or a recombinant MAGE-A3 protein vaccine with AS15. Vaccine site biopsies were obtained after one or multiple vaccines. T cells accumulated early after vaccines with AS15, but this was not durable or of the same magnitude as vaccination in IFA. Vaccines with AS15 increased durable expression of DC- and T cell-related genes, as well as PD-L1 and IDO1, suggesting complex activation and regulation of innate and adaptive immune function with AS15. These changes were generally greater with vaccines containing IFA, but IFA induced reduction in myeloid suppressor cells markers. Evidence of tertiary lymphoid structure (TLS) formation was observed with both adjuvants. Our findings highlight adjuvant-dependent changes in immune features at the VSME that may impact systemic immune responses.

TLR expression profiles are a function of disease status in rheumatoid arthritis and experimental arthritis.

The role of the innate immune system has been established in the initiation and perpetuation of inflammatory disease, but less attention has been paid to its role in the resolution of inflammation and return to homeostasis. Toll-like receptor (TLR) expression profiles were analysed in tissues with differing disease status in rheumatoid arthritis (RA), ankylosing spondylitis (AS), and in experimental arthritis. TLR gene expression was measured in whole blood and monocytes, before and after TNF blockade. In RA and osteoarthritis synovia, the expression of TLRs was quantified by standard curve qPCR. In addition, four distinct stages of disease were defined and validated in collagen-induced arthritis (CIA), the gold standard animal model for RA - pre-onset, early disease, late disease and immunised mice that were resistant to the development of disease. TLR expression was measured in spleens, lymph nodes, blood cells, liver and the paws (inflamed and unaffected). In RA whole blood, the expression of TLR1, 4 and 6 was significantly reduced by TNF blockade but the differences in TLR expression profiles between responders and non-responders were less pronounced than the differences between RA and AS patients. In RA non-responders, monocytes had greater TLR2 expression prior to therapy compared to responders. The expression of TLR1, 2, 4 and 8 was higher in RA synovium compared to control OA synovium. Circulating cytokine levels in CIA resistant mice were similar to naïve mice, but anti-collagen antibodies were similar to arthritic mice. Distinct profiles of inflammatory gene expression were mapped in paws and organs with differing disease status. TLR expression in arthritic paws tended to be similar in early and late disease, with TLR1 and 2 moderately higher in late disease. TLR expression in unaffected paws varied according to gene and disease status but was generally lower in resistant paws. Disease status-specific profiles of TLR expression were observed in spleens, lymph nodes, blood cells and the liver. Notably, TLR2 expression rose then fell in the transition from naïve to pre-onset to early arthritis. TLR gene expression profiles are strongly associated with disease status. In particular, increased expression in the blood precedes clinical manifestation.

Anticentromere antibody induced by immunization with centromere protein a and Freund's complete adjuvant may interfere with mouse oocyte meiosis.

BACKGROUND: Anticentromere antibody (ACA) is a member of the antinuclear antibody (ANA) family, and recent studies have found that ACA may be associated with oocyte maturation disorders; however, the possible mechanism behind this phenomenon remains unknown. We conducted this study to investigate whether ACA could penetrate into the living oocytes and interfere with oocyte meiosis in a mouse model. METHODS: We divided mice into three groups: human recombinant centromere protein-A (human CENP-A, HA) and complete Freund's adjuvant (CFA) were used to immunize mice for the study group (HA + CFA), and mice injected with CFA (CFA group) or saline (Saline group), respectively, served as controls. After immunization, serum anti-CENP-A antibody was detected by indirect immunofluorescence assay (IIFT) and enzyme-linked immunosorbent assay (ELISA). Chromosome alignment and intracellular IgG localization in MI- and MII-stage oocytes were investigated by immunofluorescence analysis. RESULTS: Positive ACAs were successfully induced by immunization with CENP-A and CFA, and results showed that the serum level of anti-CENP-A antibody was significantly higher in the HA + CFA group compared with the control groups. There was marked increase of chromosome misalignments in MI and MII oocytes in the HA + CFA group compared to the control groups. However, no oocytes from any of the three groups showed intracellular antibody immunofluorescence. CONCLUSIONS: The development and maturation of oocytes were impaired in peripheral ACA positive mice, which exhibited severe chromosomal misalignments in metaphase meiosis; however, no evidence of ACAs entering the oocytes was observed, thus the underlying mechanism needs further exploration.

Effect of the Fungal Immunomodulatory Protein FIP-fve in the Neutrophilic Asthma Animal Model.

BACKGROUND: Asthma animal models provide valuable information about the pathogenesis and the treatment of asthma. An ovalbumin (OVA)/complete Freund's adjuvant (CFA)-sensitized model was developed to induce neutrophil-dominant asthma and to investigate whether fungal immunomodulatory peptide-fve (FIP-fve) could improve asthma features in the OVA/CFA-sensitized model. METHODS: We used female BALB/c mice and sensitized them intraperitoneally with OVA/CFA on days 1, 2, and 3. On days 14, 17, 21, 24, and 27, they were challenged with intranasal OVA. The airway hyper-responsiveness (AHR) was detected by BUXCO, inflammatory cells were stained with Liu's stain, the cytokines were detected using ELISA, and the airway inflammation was analyzed with hematoxylin and eosin stain. RESULTS: According to the results, OVA/CFA sensitization could induce AHR, high levels of IgE, and inflammatory cells especially neutrophils infiltration in the lung and airway inflammation. IL-4, IL-5, IL-6, IL-8, IL-10, IL-13, IL-17, IL-25, IL-33, and transforming growth factor-ß (TGF-ß) increased in the OVA/CFA-sensitized mice. OVA/CFA-sensitized mice treated with FIP-fve not only increased IL-12 and IFN-γ but also decreased IL-4, IL-5, IL-6, IL-8, IL-13, IL-17, IL-25, IL-33, and TGF-ß in the bronchoalveolar lavage fluid. Moreover, FIP-fve significantly decreased neutrophil infiltration in the lung. CONCLUSION: The OVA/CFA model induced neutrophilic asthma successfully, and FIP-fve improved neutrophil-dominant asthma.

Thymic stromal lymphopoietin is a key cytokine for the immunomodulation of atherogenesis with Freund's adjuvant.

Adaptive immune responses regulate the development of atherosclerosis, with a detrimental effect of type 1 but a protective role of type 2 immune responses. Immunization of Apolipoprotein E-deficient (ApoE-/- ) mice with Freund's adjuvant inhibits the development of atherosclerosis. However, the underlying mechanisms are not fully understood. Thymic stromal lymphopoietin (TSLP) is an IL7-like cytokine with essential impact on type 2 immune responses (Th2). Thymic stromal lymphopoietin is strongly expressed in epithelial cells of the skin, but also in various immune cells following appropriate stimulation. In this study, we investigated whether TSLP may be crucial for the anti-atherogenic effect of Freund's adjuvant. Subcutaneous injection of complete Freund's adjuvant (CFA) rapidly led to the expression of TSLP and IL1ß at the site of injection. In male mice, CFA-induced TSLP occurred in immigrated monocytes-and not epithelial cells-and was dependent on NLRP3 inflammasome activation and IL1ß-signalling. In females, CFA-induced TSLP was independent of IL1ß and upon ovariectomy. CFA/OVA led to a more pronounced imbalance of the T cell response in TSLPR-/- mice, with increased INFγ/IL4 ratio compared with wild-type controls. To test whether TSLP contributes to the anti-atherogenic effects of Freund's adjuvant, we treated ApoE-/- and ApoE-/- /TSLPR-/- mice with either CFA/IFA or PBS. ApoE-/- mice showed less atherogenesis upon CFA/IFA compared with PBS injections. ApoE-/- /TSLPR-/- mice had no attenuation of atherogenesis upon CFA/IFA treatment. Freund's adjuvant executes significant immune-modulating effects via TSLP induction. TSLP-TSLPR signalling is critical for CFA/IFA-mediated attenuation of atherosclerosis.

The adjuvant effect of melanin is superior to incomplete Freund's adjuvant in subunit/peptide vaccines in mice.

Peptide vaccines represent an attractive alternative to conventional anti-tumor therapies, but have not yet achieved significant clinical efficacy with commonly used formulations. Combination of short antigenic peptides, synthetic melanin and TLR9 agonist (Toll-like receptor 9, CpG-28) was reported as highly efficient to trigger strong CD8 + T-cell responses. We compared this vaccine approach to the standard adjuvant formulation that combines the incomplete Freund's adjuvant (IFA) and CpG-28, using either an ovalbumin epitope (pOVA30) or a spontaneously occurring tumor neoepitope (mAdpgk).Melanin-based vaccine induced significantly higher cytotoxic T lymphocytes (CTL) responses than IFA-based vaccine in both pOVA30- and mAdpgk-targeted vaccines. The anti-tumor efficacy of melanin-based vaccine was further assessed in mice, grafted either with E.G7-OVA cells (E.G7 cells transfected with ovalbumin) or with MC38 cells that spontaneously express the mAdpgk neoepitope. Melanin-based vaccine induced a major inhibition of E.G7-OVA tumor growth when compared to IFA-based vaccine (p < 0.001), but tumors eventually relapsed from day 24. In the MC38 tumor model, no significant inhibition of tumor growth was observed. In both cases, tumor escape appeared related to the loss of antigen presentation by tumor cells (loss of ovalbumin expression in E.G7-OVA model; poor presentation of mAdpgk in MC38 model), although the CTL responses displayed an effector memory phenotype, a high cytolytic potential and low programmed cell death-1 (PD1) expression.In conclusion, synthetic melanin can be efficiently used as an adjuvant to enhance T-cells response against subunit vaccine antigens and compared favorably to the classic combination of IFA and TLR9 agonist in mice.

VISTA deficiency protects from immune complex-mediated glomerulonephritis by inhibiting neutrophil activation.

V-type immunoglobulin domain-containing suppressor of T-cell activation (VISTA) is a negative checkpoint regulator of T cells. We assessed VISTA deficient mice in the murine nephrotoxic nephritis models of acute and chronic immune-complex mediated glomerulonephritis. We show that VISTA deficiency protects from crescentic glomerulonephritis, with no effect on the nephritogenic adaptive immune response. The early neutrophil influx was unaffected but proteinuria was reduced suggesting a reduction in neutrophil activation. In vivo, there was reduced neutrophil degranulation in VISTA deficienct mice and, in vitro, VISTA-deficient neutrophils had an impaired response to immune complexes but not to fMLP or PMA. Mice with a genetic deficiency of neutrophils due to myeloid-specific deletion of myeloid cell leukemia 1 (Mcl-1) were also protected from crescentic glomerulonephritis, indicating an essential role for neutrophils. Therefore, VISTA deficiency inhibits neutrophil activation by immune complexes and neutrophil-dependent crescentic glomerulonephritis. This suggests that VISTA is a therapeutic target for inflammatory disease. However, this would need to be balanced against a potential enhancing effect on autoimmunity.

Cordycepin exhibits a suppressive effect on T cells through inhibiting TCR signaling cascade in CFA-induced inflammation mice model.

Objective: Cordycepin has been shown to exhibit multiple pharmacological activities, such as antitumor, antifungi, antivirus, and immune-regulation activities, and is involved in the regulation of T cells. However, cordycepin that affects T cell activity is still not clear, and the molecular mechanism of cordycepin in regulation of TCR signaling has not yet been elucidated. In this study, the potential effect of cordycepin on T cells was observed in CFA-induced inflammation mice model, and the function of cordycepin in regulating TCR signaling cascade was investigated.Methods: A CFA-induced inflammation mice model was established for observing the effect of cordycepin on the thymus and spleen swellings, and T cell infiltration in paw tissue was detected by immunohistochemistry. The protein expression or phosphorilation was detected by western blotting, and the NFAT1 nuclear translocation was determined by fluorescence imaging. The cell proliferation, apoptosis, and IL-2 production were analyzed by CCK-8 method, flow cytometry, and ELISA.Results: In the mice model, the thymus and spleen swellings were suppressed and the T cell infiltration in paw tissue was inhibited by cordycepin at a concentration of 10 mg/kg. Although the expressions of ZAP70 and PLCγ1 were not significantly changed in the human T cell line Jurkat with cordycepin pretreatment, the CD3-antibody-induced phosphorylations of ZAP70 and PLCγ1 were markedly blocked. The protein level of p85 decreased when Jurkat cells were pretreated with cordycepin, and cordycepin blocked TCR downstream molecule Erk phosphorylation and NFAT1 nuclear translocation. Further investigation revealed that cordycepin inhibited T cell proliferation, reduced IL-2 production, and induced T cell apoptosis. Conclusions: These findings suggest that cordycepin regulates TCR signaling to inhibit excessive T cell activation in inflammation. Thus, cordycepin may be a potential therapeutic application in inflammation-associated diseases.

A clinically applicable adjuvant for an atherosclerosis vaccine in mice.

Vaccination with MHC-II-restricted peptides from Apolipoprotein B (ApoB) with complete and incomplete Freund's adjuvant (CFA/IFA) is known to protect mice from atherosclerosis. This vaccination induces antigen-specific IgG1 and IgG2c antibody responses and a robust CD4 T cell response in lymph nodes. However, CFA/IFA cannot be used in humans. To find a clinically applicable adjuvant, we tested the effect of vaccinating Apoe-deficient mice with ApoB peptide P6 (TGAYSNASSTESASY). In a broad screening experiment, Addavax, a squalene-based oil-in-water adjuvant similar to MF59, was the only adjuvant that showed similar efficacy as CFA/IFA. This was confirmed in a confirmation experiment for both the aortic arch and whole aorta analyzed by en face analysis after atherosclerotic lesion staining. Mechanistically, restimulated peritoneal cells from mice immunized with P6 in Addavax released significant amounts of IL-10. Unlike P6 in CFA/IFA, vaccination with P6 in Addavax did not induce any detectable IgG1 or IgG2c antibodies to P6. These data suggest that squalene-based adjuvants such as MF59 are good candidate adjuvants for developing a clinically effective atherosclerosis vaccine.

Critical role of RAGE and HMGB1 in inflammatory heart disease.

Autoimmune response to cardiac troponin I (TnI) induces inflammation and fibrosis in the myocardium. High-mobility group box 1 (HMGB1) is a multifunctional protein that exerts proinflammatory activity by mainly binding to receptor for advanced glycation end products (RAGE). The involvement of the HMGB1-RAGE axis in the pathogenesis of inflammatory cardiomyopathy is yet not fully understood. Using the well-established model of TnI-induced experimental autoimmune myocarditis (EAM), we demonstrated that both local and systemic HMGB1 protein expression was elevated in wild-type (wt) mice after TnI immunization. Additionally, pharmacological inhibition of HMGB1 using glycyrrhizin or anti-HMGB1 antibody reduced inflammation in hearts of TnI-immunized wt mice. Furthermore, RAGE knockout (RAGE-ko) mice immunized with TnI showed no structural or physiological signs of cardiac impairment. Moreover, cardiac overexpression of HMGB1 using adeno-associated virus (AAV) vectors induced inflammation in the hearts of both wt and RAGE-ko mice. Finally, patients with myocarditis displayed increased local and systemic HMGB1 and soluble RAGE (sRAGE) expression. Together, our study highlights that HMGB1 and its main receptor, RAGE, appear to be crucial factors in the pathogenesis of TnI-induced EAM, because inhibition of HMGB1 and ablation of RAGE suppressed inflammation in the heart. Moreover, the proinflammatory effect of HMGB1 is not necessarily dependent on RAGE only. Other receptors of HMGB1 such as Toll-like receptors (TLRs) may also be involved in disease pathogenesis. These findings could be confirmed by the clinical relevance of HMGB1 and sRAGE. Therefore, blockage of one of these molecules might represent a novel therapeutic strategy in the treatment of autoimmune myocarditis and inflammatory cardiomyopathy.

Adjuvant Potential of Poly-α-l-Glutamine from the Cell Wall of Mycobacterium tuberculosis.

Novel adjuvants are in demand for improving the efficacy of human vaccines. The immunomodulatory properties of Mycobacterium tuberculosis cell wall components have been highlighted in the formulation of complete Freund's adjuvant (CFA). We have explored the adjuvant potential of poly-α-l-glutamine (PLG), a lesser-known constituent of the pathogenic mycobacterial cell wall. Immune parameters indicated that the adjuvant potency of PLG was statistically comparable to that of CFA and better than that of alum in the context of H1 antigen (Ag85B and ESAT-6 fusion). At 1 mg/dose, PLG augmented the immune response of Ag85B, BP26, and protective antigen (PA) by increasing serum antibodies and cytokines in the culture supernatant of antigen-stimulated splenocytes. PLG modulated the humoral response of vaccine candidate ESAT-6, eliciting significantly higher levels of total IgG and isotypes (IgG1, IgG2a, and IgG2b). Additionally, the splenocytes from PLG-adjuvanted mice displayed a robust increase in the Th1-specific gamma interferon, tumor necrosis factor alpha, interleukin-2 (IL-2), Th2-specific IL-6 and IL-10, and Th17-specific IL-17A cytokines upon antigenic stimulation. PLG improved the protective efficacy of ESAT-6 by reducing bacillary load in the lung and spleen as well as granuloma formation, and it helped in maintaining vital health parameters of mice challenged with M. tuberculosis The median survival time of PLG-adjuvanted mice was 205 days, compared to 146 days for dimethyl-dioctadecyl ammonium bromide-monophosphoryl lipid A (DDA-MPL)-vaccinated groups and 224 days for Mycobacterium bovis BCG-vaccinated groups. PLG enhanced the efficiency of the ESAT-6 vaccine to the level of BCG and better than that of DDA-MPL (P < 0.05), with no ill effect in C57BL/6J mice. Our results propose that PLG is a promising adjuvant candidate for advanced experimentation.

Paeoniflorin Attenuates Inflammatory Pain by Inhibiting Microglial Activation and Akt-NF-κB Signaling in the Central Nervous System.

BACKGROUND/AIMS: Paeoniflorin (PF) is known to have anti-inflammatory and paregoric effects, but the mechanism underlying its analgesic effect remains unclear. The aim of this study was to clarify the effect of PF on Freund's complete adjuvant (CFA)-induced inflammatory pain and explore the underlying molecular mechanism. METHODS: An inflammatory pain model was established by intraplantar injection of CFA in C57BL/6J mice. After intrathecal injection of PF daily for 8 consecutive days, thermal and mechanical withdrawal thresholds, the levels of inflammatory factors TNF-α, IL-1ß and IL-6, microglial activity, and the expression of Akt-NF-κB signaling pathway in the spinal cord tissue were detected by animal ethological test, cell culture, enzyme-linked immunosorbent assay, immunofluorescence histochemistry, and western blot. RESULTS: PF inhibited the spinal microglial activation in the CFA-induced pain model. The production of proinflammatory cytokines was decreased in the central nervous system after PF treatment both in vivo and in vitro. PF further displayed a remarkable effect on inhibiting the activation of Akt-NF-κB signaling pathway in vivo and in vitro. CONCLUSION: These results suggest that PF is a potential therapeutic agent for inflammatory pain and merits further investigation.

Immunization with the outer membrane proteins OmpK17 and OmpK36 elicits protection against Klebsiella pneumoniae in the murine infection model.

Klebsiella pneumoniae is a Gram-negative bacterium that is increasingly reported as a serious nosocomial and community-acquired pathogen. In the current study, two K. pneumoniae antigens, OmpK17 and OmpK36, as well as their fusion protein cognate F36/17 were investigated as potential vaccine candidates in a murine infection model. Three immunoadjuvants, namely the Gram-positive Enhancer Matrix (GEM) adjuvant, synthetic hemozoin (Hz) adjuvant and incomplete Freund's adjuvant (IFA) were evaluated. Genes of OmpK17 and OmpK36 antigens as well as their fusion protein were cloned in Escherichia coli for recombinant expression. Mice were immunized thrice with the individual recombinant purified antigens adjuvanted with one of the three adjuvants. Two weeks after the last booster, animals were challenged with a lethal dose of K. pneumoniae and immune protection parameters were assessed. Animals immunized with GEM- or Hz-adjuvanted K. pneumoniae antigens did not show significant protection upon bacterial challenge. Animals immunized with subcutaneous IFA-adjuvanted antigens showed the best results with survival percentages of 50, 60 and 50% for groups immunized with OmpK17, OmpK36 and F36/17, respectively. Serum IgG1, rather than IgG2a, antibodies were the most prevalent following vaccination indicating bias towards T helper type 2 (Th2) immune response. Opsonophagocytic assays demonstrated significant percentage killing in case of animals immunized with IFA-adjuvanted antigens. Overall, OmpK17 and OmpK36 are promising vaccine antigens which are worthy of further optimization of the immunization conditions, particularly the used immunoadjuvants, in order to achieve full protection against K. pneumoniae.

Effect of Electroacupuncture in "Zusanli" and "Kunlun" Acupoints on TLR4 Signaling Pathway of Adjuvant Arthritis Rats.

BACKGROUND: Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease. Previous study suggested that toll-like receptor (TLR) signaling pathway contributes to the development and progression of RA. In recent years, acupuncture has become one of the most vital treatments of arthralgia. But little is known about the mechanisms of improving RA by acupuncture. STUDY QUESTION: The study studied the effect of electroacupuncture in "Zusanli" and "Kunlun" acupoints on the expression of TLR4, myeloid differentiation factor 88 (MYD88), and NF-κB in adjuvant arthritis rats to clarify the molecular mechanism of acupuncture of RA. STUDY DESIGN: A rat model of adjuvant arthritis was established with injection of 0.1 mL Freund complete adjuvant in the right hindlimb footpad. We next punctured the Zusanli and Kunlun acupoints with 0.25 × 40-mm acupuncture needles to 5-mm depth. Then, we performed electroacupuncture treatment for 28 days with frequency of 2 Hz and intensity of 2 mA, once a day and 30 minutes each time. MEASURES AND OUTCOMES: Arthritis index and paw swelling were measured every week. FQ-PCR and western blot were used to detect the expression of TLR4, MYD88, and NF-κB. RESULTS: Paw swelling of rats injected with Freund complete adjuvant was more serious than that of the normal rats, which illustrated the successful establishment of adjuvant arthritis rat model. After treatment for 14 days, the paw swelling and joint symptoms score decreased, paw tissue inflammation eased in the rats of treatment group compared with the model group during the same period. After treatment for 28 days, the expression of TLR4, MYD88, and NF-κB in the ankle bone tissues decreased at both mRNA and protein levels. CONCLUSIONS: Stimulation with electric needle in Zusanli and Kunlun acupoints can reduce the expression of TLR4, MYD88, and NF-κB, which play an important role in treatment of adjuvant arthritis.

Bcl-2 inhibitors reduce steroid-insensitive airway inflammation.

BACKGROUND: Asthmatic inflammation is dominated by accumulation of either eosinophils, neutrophils, or both in the airways. Disposal of these inflammatory cells is the key to disease control. Eosinophilic airway inflammation is responsive to corticosteroid treatment, whereas neutrophilic inflammation is resistant and increases the burden of global health care. Corticosteroid-resistant neutrophilic asthma remains mechanistically poorly understood and requires novel effective therapeutic strategies. OBJECTIVE: We sought to explore the underlying mechanisms of airway inflammation persistence, as well as corticosteroid resistance, and to investigate a new strategy of effective treatment against corticosteroid-insensitive neutrophilic asthma. METHODS: Mouse models of either eosinophil-dominated or neutrophil-dominated airway inflammation were used in this study to test corticosteroid sensitivity in vivo and in vitro. We also used vav-Bcl-2 transgenic mice to confirm the importance of granulocytes apoptosis in the clearance of airway inflammation. Finally, the Bcl-2 inhibitors ABT-737 or ABT-199 were tested for their therapeutic effects against eosinophilic or neutrophilic airway inflammation and airway hyperresponsiveness. RESULTS: Overexpression of Bcl-2 protein was found to be responsible for persistence of granulocytes in bronchoalveolar lavage fluid after allergic challenge. This was important because allergen-induced airway inflammation aggravated and persisted in vav-Bcl-2 transgenic mice, in which nucleated hematopoietic cells were overexpressed with Bcl-2 and resistant to apoptosis. The Bcl-2 inhibitors ABT-737 or ABT-199 play efficient roles in alleviation of either eosinophilic or corticosteroid-resistant neutrophilic airway inflammation by inducing apoptosis of immune cells, such as eosinophils, neutrophils, TH2 cells, TH17 cells, and dendritic cells. Moreover, these inhibitors were found to be more efficient than steroids to induce granulocyte apoptosis ex vivo from patients with severe asthma. CONCLUSION: Apoptosis of inflammatory cells is essential for clearance of allergen-induced airway inflammation. The Bcl-2 inhibitors ABT-737 or ABT-199 might be promising drugs for the treatment of airway inflammation, especially for corticosteroid-insensitive neutrophilic airway inflammation.

Preclinical Explorative Assessment of Celecoxib-Based Biocompatible Lipidic Nanocarriers for the Management of CFA-Induced Rheumatoid Arthritis in Wistar Rats.

Celecoxib (CXB), a COX-2 inhibitor, is primarily indicated for long-term treatment of rheumatoid arthritis (RA). The effective therapeutic efficacy of CXB on RA via oral administration shows adverse systemic complications, and therefore, local application of CXB has been recommended. The aim of the present study was to develop and characterize solid lipid nanoparticles (SLNs) with enhanced skin permeation potential of CXB. The particle size, polydispersity index (PDI), and percentage drug entrapment (PDE) of the developed SLNs (CXB-SLNs) were found to be 240 nm, < 0.3, and ~ 86% respectively. The developed SLNs exhibited sustained release up to 70% at the end of 48 h. Drug permeation was found to be 45% for SLN gel and 31% for conventional gel. The dermatokinetic studies also confirmed enhanced permeation of CXB in the epidermis and dermis and revealed superiority of the developed SLN gel vis-à-vis the conventional gel. Further, in the CFA-induced arthritis rat model, % arthritis index (AI) of the CXB-SLN gel formulation was found to be very less (18.54%) as compared to untreated (187.34%) and conventional gel-treated (91.61%) animals. In conclusion, the current study can provide a suitable alternative for the development of an effective topical formulation of CXB in lipid nanocarriers.

Cytotoxic T cells modulate inflammation and endogenous opioid analgesia in chronic arthritis.

BACKGROUND: This study examined the development of chronic pain, a cardinal symptom of rheumatoid arthritis (RA), in mice with antigen- and collagen-induced arthritis (ACIA). Since the role of CD8+ T cells in arthritis is controversial, we investigated the consequences of CD8-depletion on arthritis development and opioid modulation of pain in this novel model of chronic autoimmune arthritis. METHODS: Disease severity in control and CD8-depleted animals was determined by histological assessment of knee-joint sections and measurement of autoantibody formation. Pain was evaluated by measuring mechanical allodynia and thermal hyperalgesia in von Frey and Hargreaves tests, respectively. The production and release of endogenous opioids and inflammatory cytokines was assessed in immunoassays. RESULTS: In ACIA, mice display persistent mechanical allodynia and thermal hyperalgesia for more than 2 months after induction of arthritis. The blockade of peripheral opioid receptors with naloxone-methiodide (NLXM) transiently increased thermal hyperalgesia, indicating that endogenous opioid peptides were released in the arthritic joint to inhibit pain. CD8+ T cell depletion did not affect autoantibody formation or severity of joint inflammation, but serum levels of the pro-inflammatory cytokines TNFα and IL-17 were increased. The release of opioid peptides from explanted arthritic knee cells and the NLXM effect were significantly reduced in the absence of CD8+ T cells. CONCLUSIONS: We have successfully modeled the development of chronic pain, a hallmark of RA, in ACIA. Furthermore, we detected a yet unknown protective role of CD8+ T cells in chronic ACIA since pro-inflammatory cytokines rose and opioid peptide release decreased in the absence of these cells.

Lack of Fc Gamma Receptor IIIA Promotes Rather than Suppresses Humoral and Cellular Immune Responses after Mucosal or Parenteral Immunization with Antigen and Adjuvants.

The Fcγ receptor IIIA (FcγRIIIA) has traditionally been known as a positive regulator of immune responses. Consistent with this, mice deficient in FcγRIIIA are protected from various inflammation-associated pathologies including several autoimmune diseases. In contrast to this accepted dogma, we show here that mice lacking FcγRIIIA developed increased rather than reduced both humoral and cellular immune responses to mucosal (sublingual) immunization with ovalbumin (OVA) given together with the strong mucosal adjuvant cholera toxin as well as to parenteral (subcutaneous) immunization with OVA in complete Freund's adjuvant. After either route of immunization, in comparison with concomitantly immunized wild-type mice, FcγRIIIA-/- mice had increased serum anti-OVA IgG (IgG1 but not IgG2) antibody responses as well as augmented cellular responses that included memory B cells and effector T cells. The increments in immune responses in FcγRIIIA-/- mice were similar to those seen in FcγRIIB-/- mice. Furthermore, OVA-pulsed FcγRIIIA-/- DCs, similar to OVA-specific FcγRIIB-/- DCs, had enhanced capacity to activate OVA-specific OT-II T cells, which was even further pronounced when DCs were pulsed with IgG1-complexed OVA. Our data support an inhibitory-regulatory role of FcγRIIIA on vaccine/adjuvant-induced immune responses and demonstrate that lack of FcγRIIIA can promote rather than suppress both humoral and cellular immune responses.

Endogenous adaptation to low oxygen modulates T-cell regulatory pathways in EAE.

BACKGROUND: In the brain, chronic inflammatory activity may lead to compromised delivery of oxygen and glucose suggesting that therapeutic approaches aimed at restoring metabolic balance may be useful. In vivo exposure to chronic mild normobaric hypoxia (10 % oxygen) leads to a number of endogenous adaptations that includes vascular remodeling (angioplasticity). Angioplasticity promotes tissue survival. We have previously shown that induction of adaptive angioplasticity modulates the disease pattern in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE). In the present study, we define mechanisms by which adaptation to low oxygen functionally ameliorates the signs and symptoms of EAE and for the first time show that tissue hypoxia may fundamentally alter neurodegenerative disease. METHODS: C57BL/6 mice were immunized with MOG, and some of them were kept in the hypoxia chambers (day 0) and exposed to 10 % oxygen for 3 weeks, while the others were kept at normoxic environment. Sham-immunized controls were included in both hypoxic and normoxic groups. Animals were sacrificed at pre-clinical and peak disease periods for tissue collection and analysis. RESULTS: Exposure to mild hypoxia decreased histological evidence of inflammation. Decreased numbers of cluster of differentiation (CD)4+ T cells were found in the hypoxic spinal cords associated with a delayed Th17-specific cytokine response. Hypoxia-induced changes did not alter the sensitization of peripheral T cells to the MOG peptide. Exposure to mild hypoxia induced significant increases in anti-inflammatory IL-10 levels and an increase in the number of spinal cord CD25+FoxP3+ T-regulatory cells. CONCLUSIONS: Acclimatization to mild hypoxia incites a number of endogenous adaptations that induces an anti-inflammatory milieu. Further understanding of these mechanisms system may pinpoint possible new therapeutic targets to treat neurodegenerative disease.