ABSTRACT
Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.
Subject(s)
Cell Adhesion Molecules/metabolism , Endocytosis , Epithelial Cells/metabolism , Measles virus/metabolism , Nectins/metabolism , Virus Internalization , Biological Transport, Active/genetics , Cell Adhesion/genetics , Cell Adhesion Molecules/genetics , Cell Line , Humans , Measles virus/genetics , Nectins/geneticsABSTRACT
A Salmonella therapeutic expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) fimbriae protects against collagen-induced arthritis (CIA) by eliciting two regulatory T cell (Treg) subsets: TGF-ß-producing Foxp3(-)CD39(+)CD4(+) T cells and IL-10-producing Foxp3(+)CD39(+)CD4(+) T cells. However, it is unclear whether CFA/I fimbriae alone are protective and whether other regulatory cytokines are involved, especially in the context for the EBI3-sharing cytokines, Treg-derived IL-35 and APC-derived IL-27, both capable of suppressing Th17 cells and regulating autoimmune diseases. Subsequent evaluation revealed that a single oral dose of purified, soluble CFA/I fimbriae protected against CIA as effectively as did Salmonella-CFA/I and found that Foxp3(+)CD39(+)CD4(+) T cells were the source of secreted IL-35, whereas IL-27 production by CD11c(+) cells was inhibited. Inquiring into their relevance, CFA/I fimbriae-treated IL-27R-deficient (WSX-1(-/-)) mice were equally protected against CIA as were wild-type mice, suggesting a limited role for IL-27. In contrast, CFA/I fimbriae-mediated protection was abated in EBI3(-/-) mice, accompanied by the loss of TGF-ß- and IL-10-producing Tregs. Adoptive transfer of C57BL/6 CD39(+)CD4(+) T cells to EBI3(-/-) mice with concurrent CFA/I plus IL-35 treatment effectively stimulated Tregs suppressing proinflammatory collagen II-specific Th cells. In contrast, recipients cotransferred with C57BL/6 and EBI3(-/-) CD39(+)CD4(+) T cells and treated with CFA/I plus IL-35 were not protected, implicating the importance of endogenous IL-35 for conferring CFA/I-mediated protection. Thus, CFA/I fimbriae stimulate IL-35 required for the coinduction of TGF-ß and IL-10.
Subject(s)
Arthritis, Experimental/immunology , Escherichia coli/immunology , Fimbriae Proteins/immunology , Fimbriae, Bacterial/immunology , Interleukin-27/immunology , Interleukins/immunology , Animals , Antigens, Bacterial/immunology , Antigens, CD/immunology , Arthritis, Experimental/chemically induced , Forkhead Transcription Factors/immunology , Interleukin-10/immunology , Male , Mice , Mice, Inbred C57BL , Minor Histocompatibility Antigens , Receptors, Cytokine/immunology , Th17 Cells/immunology , Transforming Growth Factor beta/immunologyABSTRACT
HIV infection results in a complex immunodeficiency due to loss of CD4(+) T cells, impaired type I interferon (IFN) responses, and B cell dysfunctions causing susceptibility to opportunistic infections such as Pneumocystis murina pneumonia and unexplained comorbidities, including bone marrow dysfunctions. Type I IFNs and B cells critically contribute to immunity to Pneumocystis lung infection. We recently also identified B cells as supporters of on-demand hematopoiesis following Pneumocystis infection that would otherwise be hampered due to systemic immune effects initiated in the context of a defective type I IFN system. While studying the role of type I IFNs in immunity to Pneumocystis infection, we discovered that mice lacking both lymphocytes and type I IFN receptor (IFrag(-/-)) developed progressive bone marrow failure following infection, while lymphocyte-competent type I IFN receptor-deficient mice (IFNAR(-/-)) showed transient bone marrow depression and extramedullary hematopoiesis. Lymphocyte reconstitution of lymphocyte-deficient IFrag(-/-) mice pointed to B cells as a key player in bone marrow protection. Here we define how B cells protect on-demand hematopoiesis following Pneumocystis lung infection in our model. We demonstrate that adoptive transfer of B cells into IFrag(-/-) mice protects early hematopoietic progenitor activity during systemic responses to Pneumocystis infection, thus promoting replenishment of depleted bone marrow cells. This activity is independent of CD4(+) T cell help and B cell receptor specificity and does not require B cell migration to bone marrow. Furthermore, we show that B cells protect on-demand hematopoiesis in part by induction of interleukin-10 (IL-10)- and IL-27-mediated mechanisms. Thus, our data demonstrate an important immune modulatory role of B cells during Pneumocystis lung infection that complement the modulatory role of type I IFNs to prevent systemic complications.
Subject(s)
B-Lymphocytes/immunology , Hematopoiesis/immunology , Pneumocystis/immunology , Pneumonia, Pneumocystis/immunology , Receptor, Interferon alpha-beta/genetics , Adoptive Transfer , Anemia, Aplastic , Animals , B-Lymphocytes/transplantation , Bone Marrow Diseases , Bone Marrow Failure Disorders , CD4-Positive T-Lymphocytes/immunology , Hemoglobinuria, Paroxysmal/immunology , Hemoglobinuria, Paroxysmal/microbiology , Interferon Type I/genetics , Interleukin-10/biosynthesis , Interleukin-10/immunology , Interleukins/biosynthesis , Interleukins/immunology , Lung/immunology , Lung/microbiology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, SCIDABSTRACT
c-Jun N-terminal kinases (JNKs) participate in many physiologic and pathologic processes, including inflammatory diseases. We recently synthesized the sodium salt of IQ-1S (11H-indeno[1,2-b]quinoxalin-11-one oxime) and demonstrated that it is a high-affinity JNK inhibitor and inhibits murine delayed-type hypersensitivity. Here we show that IQ-1S is highly specific for JNK and that its neutral form is the most abundant species at physiologic pH. Molecular docking of the IQ-1S syn isomer into the JNK1 binding site gave the best pose, which corresponded to the position of cocrystallized JNK inhibitor SP600125 (1,9-pyrazoloanthrone). Evaluation of the therapeutic potential of IQ-1S showed that it inhibited matrix metalloproteinase 1 and 3 gene expression induced by interleukin-1ß in human fibroblast-like synoviocytes and significantly attenuated development of murine collagen-induced arthritis (CIA). Treatment with IQ-1S either before or after induction of CIA resulted in decreased clinical scores, and joint sections from IQ-1S-treated CIA mice exhibited only mild signs of inflammation and minimal cartilage loss compared with those from control mice. Collagen II-specific antibody responses were also reduced by IQ-1S treatment. By contrast, the inactive ketone derivative 11H-indeno[1,2-b]quinoxalin-11-one had no effect on CIA clinical scores or collagen II-specific antibody titers. IQ-1S treatment also suppressed proinflammatory cytokine and chemokine levels in joints and lymph node cells. Finally, treatment with IQ-1S increased the number of Foxp3(+)CD4(+)CD25(+) regulatory T cells in lymph nodes. Thus, IQ-1S can reduce inflammation and cartilage loss associated with CIA and can serve as a small-molecule modulator for mechanistic studies of JNK function in rheumatoid arthritis.
Subject(s)
Arthritis, Experimental/drug therapy , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Oximes/pharmacology , Protein Kinase Inhibitors/therapeutic use , Quinoxalines/pharmacology , Animals , Antibodies/analysis , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , Binding Sites/drug effects , Cytokines/metabolism , Dose-Response Relationship, Drug , Joints/pathology , Male , Matrix Metalloproteinases/biosynthesis , Mice , Mice, Inbred C57BL , Models, Molecular , Oximes/therapeutic use , Quinoxalines/therapeutic use , Synovial Fluid/cytology , Synovial Fluid/metabolism , T-Lymphocytes, Regulatory/drug effectsABSTRACT
Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-γ via enhanced IL-4, IL-10, and TGF-ß. To identify the responsible regulatory CD4(+) T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39(+)CD4(+) T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39(+)CD4(+) T cells into CIA mice conferred complete protection, whereas CD39(-)CD4(+) T cells did not. Subsequent analysis of vaccinated Foxp3-GFP mice revealed the CD39(+) T cells were composed of Foxp3-GFP(-) and Foxp3-GFP(+) subpopulations. Although each adoptively transferred Salmonella-CFA/I-induced Foxp3(-) and Foxp3(+)CD39(+)CD4(+) T cells could protect against CIA, each subset was not as efficacious as total CD39(+)CD4(+) T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed Foxp3(-) CD39(+)CD4(+) T cells produced TGF-ß, and Foxp3(+)CD39(+)CD4(+) T cells produced IL-10, showing a segregation of function. Moreover, donor Foxp3-GFP(-) CD4(+) T cells converted to Foxp3-GFP(+) CD39(+)CD4(+) T cells in the recipients, showing plasticity of these regulatory T cells. TGF-ß was found to be essential for protection because in vivo TGF-ß neutralization reversed activation of CREB and reduced the development of CD39(+)CD4(+) T cells. Thus, CD39 apyrase-expressing CD4(+) T cells stimulated by Salmonella-CFA/I are composed of TGF-ß-producing Foxp3(-) CD39(+)CD4(+) T cells and support the stimulation of IL-10-producing Foxp3(+) CD39(+)CD4(+) T cells.
Subject(s)
Antigens, CD/physiology , Apyrase/physiology , Arthritis, Experimental/immunology , CD4-Positive T-Lymphocytes/immunology , Collagen/toxicity , Forkhead Transcription Factors/physiology , Interleukin-10/physiology , Transforming Growth Factor beta/biosynthesis , Animals , Antigens, CD/biosynthesis , Apyrase/biosynthesis , Arthritis, Experimental/pathology , Arthritis, Experimental/prevention & control , CD4-Positive T-Lymphocytes/metabolism , Collagen/administration & dosage , Fimbriae Proteins , Forkhead Transcription Factors/biosynthesis , Forkhead Transcription Factors/genetics , Interleukin-10/biosynthesis , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Salmonella Vaccines/administration & dosage , Salmonella Vaccines/immunology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/metabolism , T-Lymphocytes, Regulatory/transplantation , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunologyABSTRACT
In efforts to identify novel small molecules with anti-inflammatory properties, we discovered a unique series of tetracyclic indenoquinoxaline derivatives that inhibited lipopolysaccharide (LPS)-induced nuclear factor-κB/activating protein 1 activation. Compound IQ-1 (11H-indeno[1,2-b]quinoxalin-11-one oxime) was found to be a potent, noncytotoxic inhibitor of pro-inflammatory cytokine [interleukin (IL)-1α, IL-1ß, IL-6, IL-10, tumor necrosis factor (TNF)-α, interferon-γ, and granulocyte-macrophage colony-stimulating factor] and nitric oxide production by human and murine monocyte/macrophages. Three additional potent inhibitors of cytokine production were identified through further screening of IQ-1 analogs. The sodium salt of IQ-1 inhibited LPS-induced TNF-α and IL-6 production in MonoMac-6 cells with IC(50) values of 0.25 and 0.61 µM, respectively. Screening of 131 protein kinases revealed that derivative IQ-3 [11H-indeno[1,2-b]quinoxalin-11-one-O-(2-furoyl)oxime]was a specific inhibitor of the c-Jun N-terminal kinase (JNK) family, with preference for JNK3. This compound, as well as IQ-1 and three additional oxime indenoquinoxalines, were found to be high-affinity JNK inhibitors with nanomolar binding affinity and ability to inhibit c-Jun phosphorylation. Furthermore, docking studies showed that hydrogen bonding interactions of the active indenoquinoxalines with Asn152, Gln155, and Met149 of JNK3 played an important role in enzyme binding activity. Finally, we showed that the sodium salt of IQ-1 had favorable pharmacokinetics and inhibited the ovalbumin-induced CD4(+) T-cell immune response in a murine delayed-type hypersensitivity model in vivo. We conclude that compounds with an indenoquinoxaline nucleus can serve as specific small-molecule modulators for mechanistic studies of JNKs as well as a potential leads for the development of anti-inflammatory drugs.
Subject(s)
JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Oximes/pharmacology , Protein Kinase Inhibitors/pharmacology , Quinoxalines/pharmacology , Animals , Blotting, Western , Cells, Cultured , Cytokines/antagonists & inhibitors , Enzyme-Linked Immunosorbent Assay , Humans , Hydrogen Bonding , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Models, Molecular , Oximes/chemistry , Phosphorylation , Protein Kinase Inhibitors/chemistry , Quinoxalines/chemistryABSTRACT
IL-35 is produced by regulatory T cells, and this novel cytokine can downregulate Th17 cell development and inhibit autoimmune inflammation. In this work, an rIL-35, as a single-chain fusion between murine IL-12p35 and EBV-induced gene 3, was expressed in yeast. This rIL-35 inhibited OVA-specific cellular and Ab responses in OVA-challenged recipients of DO11.10 CD4+ T cells. Likewise, IL-35 inhibited clinical manifestation of collagen-induced arthritis or could cease further disease exacerbation upon initiation of IL-35 treatment. Exogenous IL-35 treatments suppressed Th1 and Th17 cells and promoted CD39 expression by CD4+ T cells. Sorted CD25-CD39+CD4+ T cells from IL-35-treated mice produced IL-10 and, upon adoptive transfer, were sufficiently potent to inhibit subsequent development of inflammation in mice with collagen-induced arthritis, whereas sorted CD25+CD39+CD4+ T cells showed reduced potency. IL-35 treatments of IL-10-/- mice failed to induce protective CD39+CD4+ T cells, demonstrating the effector role of IL-10 by IL-35 immunosuppression.
Subject(s)
Arthritis, Experimental/immunology , Immune Tolerance/immunology , Interleukin-10/immunology , Interleukin-12 Subunit p35/immunology , T-Lymphocytes, Regulatory/immunology , Adoptive Transfer , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , Apyrase/immunology , Apyrase/metabolism , Arthritis, Experimental/metabolism , Cell Separation , Flow Cytometry , Interleukin-10/biosynthesis , Interleukin-12 Subunit p35/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Transgenic , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , T-Lymphocyte Subsets/immunology , T-Lymphocyte Subsets/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection interferes with secondary viral infections within that same cell. Although SIE has been observed across many viruses, it has remained relatively understudied. A recently characterized glycoprotein D (gD)-independent SIE of alphaherpesviruses presents a novel mechanism of coinfection restriction for herpes simplex virus 1 (HSV-1) and pseudorabies virus (PRV). In this study, we evaluated the role of multiplicity of infection (MOI), receptor expression, and trafficking of virions to gain greater insight into potential mechanisms of alphaherpesvirus SIE. We observed that high-MOI secondary viral infections were able to overcome SIE in a manner that was independent of receptor availability. We next assessed virion localization during SIE through live microscopy of fluorescently labeled virions and capsid assemblies. Analysis of these fluorescent assemblies identified changes in the distribution of capsids during SIE. These results indicate that SIE during PRV infection inhibits viral entry or fusion while HSV-1 SIE inhibits infection through a postentry mechanism. Although the timing and phenotype of SIE are similar between alphaherpesviruses, the related viruses implement different mechanisms to restrict coinfection. IMPORTANCE Most viruses utilize a form of superinfection exclusion to conserve resources and control population dynamics. gD-dependent superinfection exclusion in alphaherpesviruses is well documented. However, the undercharacterized gD-independent SIE provides new insight into how alphaherpesviruses limit sequential infection. The observations described here demonstrate that gD-independent SIE differs between PRV and HSV-1. Comparing these differences provides new insights into the underlying mechanisms of SIE implemented by two related viruses.
Subject(s)
Coinfection , Herpesvirus 1, Human , Herpesvirus 1, Suid , Superinfection , Animals , VirionABSTRACT
The risk posed by Severe Acute Respiratory Syndrome Coronavirus -2 (SARS-CoV-2) dictates that live-virus research is conducted in a biosafety level 3 (BSL3) facility. Working with SARS-CoV-2 at lower biosafety levels can expedite research yet requires the virus to be fully inactivated. In this study, we validated and compared two protocols for inactivating SARS-CoV-2: heat treatment and ultraviolet irradiation. The two methods were optimized to render the virus completely incapable of infection while limiting the destructive effects of inactivation. We observed that 15 min of incubation at 65 °C completely inactivates high titer viral stocks. Complete inactivation was also achieved with minimal amounts of UV power (70,000 µJ/cm2), which is 100-fold less power than comparable studies. Once validated, the two methods were then compared for viral RNA quantification, virion purification, and antibody detection assays. We observed that UV irradiation resulted in a 2-log reduction of detectable genomes compared to heat inactivation. Protein yield following virion enrichment was equivalent for all inactivation conditions, but the quality of resulting viral proteins and virions were differentially impacted depending on inactivation method and time. Here, we outline the strengths and weaknesses of each method so that investigators might choose the one which best meets their research goals.
Subject(s)
COVID-19/virology , Disinfection/methods , SARS-CoV-2/radiation effects , Virion/radiation effects , Virus Inactivation/radiation effects , Disinfection/instrumentation , Hot Temperature , Humans , SARS-CoV-2/chemistry , SARS-CoV-2/genetics , SARS-CoV-2/physiology , Ultraviolet Rays , Viral Proteins/genetics , Viral Proteins/metabolism , Virion/chemistry , Virion/genetics , Virion/physiologyABSTRACT
Anti-inflammation immunotherapy has been successfully applied for the treatment of autoimmune diseases. Mucosal vaccines against autoimmune disorders are beneficial by influencing the regulatory compartment of gut and systemic adaptive immune systems. A Salmonella vector expressing colonization factor Ag I (CFA/I), shown to behave as an anti-inflammatory vaccine, stimulates the production of CD4(+)CD25(+) T cells and regulatory cytokines. In this work, we queried whether Salmonella-CFA/I can protect DBA/1 mice from collagen-induced arthritis. The incidence of arthritis and cartilage loss in vaccinated DBA/1 mice was remarkably lower when compared with unprotected mice. Clinical findings were accompanied by the suppression of inflammatory cytokines TNF-alpha, IL-1beta, IL-6, and IL-27. Vaccination evoked a multi-tier response consisting of IL-4 producing Th2 cells, an increased production of TGF-beta by CD4(+) T cells, and suppression of collagen II-specific CD4(+) T cell proliferation. To assess the contribution of Salmonella-CFA/I-primed CD4(+) T cells, adoptive transfer studies with total CD4(+), CD4(+)CD25(-), or CD4(+)CD25(+) T cells were performed 15 days postchallenge. Mice receiving either subset showed reduced disease incidence and low clinical scores; however, mice receiving total CD4(+) T cells showed delayed disease onset by 10 days with reduced clinical scores, reduced IL-17 and IL-27, but enhanced IL-4, IL-10, IL-13, and TGF-beta. Inhibition of TGF-beta or IL-4 compromised protective immunity. These data show that Salmonella-CFA/I vaccination of DBA/1 mice protects against collagen-induced arthritis by stimulating TGF-beta- and IL-4-producing regulatory CD4(+) T cells.
Subject(s)
Arthritis, Experimental/immunology , Arthritis, Experimental/prevention & control , Autoantigens/physiology , Collagen Type II/immunology , Salmonella Vaccines/immunology , T-Lymphocytes, Regulatory/transplantation , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Administration, Oral , Adoptive Transfer , Animals , Anti-Inflammatory Agents, Non-Steroidal/administration & dosage , Anti-Inflammatory Agents, Non-Steroidal/immunology , Arthritis, Experimental/pathology , Fimbriae Proteins/administration & dosage , Fimbriae Proteins/genetics , Fimbriae Proteins/immunology , Male , Mice , Mice, Inbred DBA , Salmonella Vaccines/administration & dosage , Salmonella Vaccines/genetics , T-Lymphocytes, Regulatory/immunology , Vaccines, DNA/geneticsABSTRACT
Intratracheal instillation of L-selectin-deficient (L-Sel(-/-)) mice with an adenovirus 2 (Ad2) vector resulted in the lack of respiratory Ad2- or beta-galactosidase-specific CTLs with concomitant long-lived beta-galactosidase transgene expression in the lungs. The absence of Ag-specific CTLs was attributed to a deficiency in lymphoid CD11c(+)CD8(+) dendritic cells (DCs) in the lower respiratory lymph nodes (LRLNs). To enable L-Sel(-/-) CTL activity, cell-sorted L-Sel(-/-)CD8(+) T cells were cocultured with cell-sorted L-Sel(+/+)CD8(+) or CD8(-) DCs or L-Sel(-/-)CD8(-) DCs. Only the CD8(+) DCs restored CTL activity; L-Sel(-/-)CD8(-) DCs failed to support L-Sel(+/+) CTLs because these remained immature, lacking the ability to express costimulatory molecules CD40, CD80, or CD86. Although no lung CD8(+) DCs were detected, the DC environment remained suppressive in L-Sel(-/-) mice evident by the lack of CTL responses following adenoviral challenge with OVA in recipient L-Sel(-/-) adoptively transferred with OT-1 CD8(+) T cells. To assess whether the L-Sel(-/-)CD8(-) DCs could be induced into maturity, microbial stimulation studies were performed showing the failure of L-Sel(-/-) LRLN to make matured DCs. When L-Sel(-/-) mice were subjected in vivo to microbial activation before Ad2 vector dosing, CTL activity was restored stimulating the renewed presence of LRLN CD8(+) DCs in L-Sel(-/-) mice. These studies show that impairment of L-Sel(-/-) DC maturation results in insufficient mature DCs that require microbial activation to restore increases in respiratory CD8(+) DCs to support CTL responses.
Subject(s)
Adenoviridae/immunology , CD8 Antigens/immunology , Dendritic Cells/immunology , L-Selectin/immunology , Lung/immunology , Lymph Nodes/immunology , T-Lymphocytes, Cytotoxic/immunology , Adenoviridae/genetics , Animals , B7-1 Antigen/immunology , B7-1 Antigen/metabolism , B7-2 Antigen/immunology , B7-2 Antigen/metabolism , CD40 Antigens/immunology , CD40 Antigens/metabolism , CD8 Antigens/metabolism , Cell Line, Tumor , Dendritic Cells/metabolism , Genetic Vectors , L-Selectin/genetics , Lipopolysaccharides/immunology , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , T-Lymphocytes, Cytotoxic/metabolismABSTRACT
Treatment with an anti-inflammatory Salmonella vaccine expressing enterotoxigenic Escherichia coli colonization factor Ag 1 (CFA/I) proved effective in stimulating protective, potent CD25(+)CD4(+) regulatory T (T(reg)) cells in susceptible mice challenged with experimental autoimmune encephalomyelitis (EAE). Because the Salmonella vector was considerably less protective, we questioned whether altering fimbrial subunit expression to resemble conventional Salmonella expression may impact T(reg) cell potency. The Salmonella-CFA/I vaccine was modified to limit fimbrial subunit expression to the intracellular compartment (Salmonella-CFA/I(IC)). SJL mice were challenged with proteolipid protein peptide 139-151 to induce EAE and orally treated with one of three Salmonella vaccines 6 days postchallenge. Treatment with Salmonella-CFA/I(IC) greatly reduced clinical disease, similarly as Salmonella-CFA/I, by subduing IL-17 and IL-21; however, mechanisms of protection differed as evident by increased IL-13 and IFN-gamma but diminished TGF-beta production by T(reg) cells from Salmonella-CFA/I(IC)-treated mice. Adoptive transfer of T(reg) cells from both CFA/I-expressing constructs was equivalent in protecting against EAE, showing minimal disease. Although not as potent in its protection, CD25(-)CD4(+) T cells from Salmonella-CFA/I(IC) showed minimal Th2 cells, but vaccination did prime these Th2 cells rendering partial protection against EAE challenge. In vivo IL-13 but not IFN-gamma neutralization compromised protection conferred by adoptive transfer with Salmonella-CFA/I(IC)-induced T(reg) cells. Thus, the Salmonella-CFA/I(IC) vaccine elicits T(reg) cells with attributes from both the Salmonella vector and Salmonella-CFA/I vaccines. Importantly, these T(reg) cells can be induced to high potency by simply vaccinating against irrelevant Ags, offering a novel approach to treat autoimmune diseases independently of the autoantigen.
Subject(s)
Cytokines/metabolism , Encephalomyelitis, Autoimmune, Experimental/immunology , Fimbriae Proteins/immunology , Interleukin-13/metabolism , Salmonella Vaccines/immunology , T-Lymphocytes, Regulatory/immunology , Th2 Cells/immunology , Adoptive Transfer , Animals , Autoantigens/immunology , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Cytokines/immunology , Encephalomyelitis, Autoimmune, Experimental/metabolism , Encephalomyelitis, Autoimmune, Experimental/prevention & control , Female , Fimbriae Proteins/metabolism , Interferon-gamma/immunology , Interferon-gamma/metabolism , Interleukin-13/immunology , Mice , T-Lymphocytes, Regulatory/metabolism , Th2 Cells/metabolismABSTRACT
IL-35, a relatively newly discovered cytokine belonging to the larger IL-12 family, shows unique anti-inflammatory properties, believed to be associated with dedicated receptors and signaling pathways. IL-35 plays a pivotal role in the development and the function of both regulatory B (Bregs) and T cells (Tregs). In order to further its therapeutic potential, a dairy Lactococcus lactis strain was engineered to express murine IL-35 (LL-IL35), and this recombinant strain was applied to suppress collagen-induced arthritis (CIA). Oral administration of LL-IL35 effectively reduced the incidence and disease severity of CIA. When administered therapeutically, LL-IL35 abruptly halted CIA progression with no increase in disease severity by reducing neutrophil influx into the joints. LL-IL35 treatment reduced IFN-γ and IL-17 3.7- and 8.5-fold, respectively, and increased IL-10 production compared to diseased mice. Foxp3+ and Foxp3- CD39+ CD4+ T cells were previously shown to be the Tregs responsible for conferring protection against CIA. Inquiry into their induction revealed that both CCR6+ and CCR6- Foxp3+or- CD39+ CD4+ T cells act as the source of the IL-10 induced by LL-IL35. Thus, this study demonstrates the feasibility and benefits of engineered probiotics for treating autoimmune diseases.
Subject(s)
Arthritis, Experimental/therapy , B-Lymphocytes, Regulatory/immunology , Interleukins/immunology , Lactococcus lactis/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/pathology , B-Lymphocytes, Regulatory/pathology , Interleukins/genetics , Lactococcus lactis/genetics , Male , Mice , T-Lymphocytes, Regulatory/pathologyABSTRACT
Autoimmune diseases arise from the loss of tolerance to self, and because the etiologies of such diseases are largely unknown, symptomatic treatments rely on anti-inflammatory and analgesic agents. Tolerogenic treatments that can reverse disease are preferred, but again, often thwarted by not knowing the responsible auto-antigens (auto-Ags). Hence, a viable alternative to stimulating regulatory T cells (Tregs) is to induce bystander tolerance. Colonization factor antigen I (CFA/I) has been shown to evoke bystander immunity and to hasten Ag-specific Treg development independent of auto-Ag. To translate in treating human autoimmune diseases, the food-based Lactococcus was engineered to express CFA/I fimbriae, and Lactococcus-CFA/I fermented milk fed to arthritic mice proved highly efficacious. Protection occurred via CD39+ Tregs producing TGF-ß and IL-10 to potently suppress TNF-α production and neutrophil influx into the joints. Thus, these data demonstrate the feasibility of oral nutraceuticals for treating arthritis, and potency of protection against arthritis was improved relative to that obtained with Salmonella-CFA/I.
Subject(s)
Antigens, CD/metabolism , Apyrase/metabolism , Arthritis, Experimental/drug therapy , Dietary Supplements , Interleukin-10/metabolism , Milk/immunology , T-Lymphocytes, Regulatory/metabolism , Transforming Growth Factor beta/metabolism , Animals , Arthritis, Experimental/immunology , Arthritis, Experimental/metabolism , Bystander Effect/immunology , Immune Tolerance/immunology , Mice , T-Lymphocytes, Regulatory/immunology , Tumor Necrosis Factor-alpha/immunologyABSTRACT
To date, efforts to treat autoimmune diseases have primarily focused on the disease symptoms rather than on the cause of the disease. In large part, this is attributed to not knowing the responsible auto-antigens (auto-Ags) for driving the self-reactivity coupled with the poor success of treating autoimmune diseases using oral tolerance methods. Nonetheless, if tolerogenic approaches or methods that stimulate regulatory T (Treg) cells can be devised, these could subdue autoimmune diseases. To forward such efforts, our approach with colonization factor antigen I (CFA/I) fimbriae is to establish bystander immunity to ultimately drive the development of auto-Ag-specific Treg cells. Using an attenuated Salmonella vaccine expressing CFA/I fimbriae, fimbriae-specific Treg cells were induced without compromising the vaccine's capacity to protect against travelers' diarrhea or salmonellosis. By adapting the vaccine's anti-inflammatory properties, it was found that it could also dampen experimental inflammatory diseases resembling multiple sclerosis (MS) and rheumatoid arthritis. Because of this bystander effect, disease-specific Treg cells are eventually induced to resolve disease. Interestingly, this same vaccine could elicit the required Treg cell subset for each disease. For MS-like disease, conventional CD25(+) Treg cells are stimulated, but for arthritis CD39(+) Treg cells are induced instead. This review article will examine the potential of treating autoimmune diseases without having previous knowledge of the auto-Ag using an innocuous antigen to stimulate Treg cells via the production of transforming growth factor-ß and interleukin-10.
Subject(s)
Antigens, Bacterial/immunology , Autoantigens/immunology , Fimbriae Proteins/immunology , Salmonella/immunology , T-Lymphocytes, Regulatory/immunology , Vaccination , Animals , Arthritis, Rheumatoid/immunology , Arthritis, Rheumatoid/prevention & control , Humans , Multiple Sclerosis/immunology , Multiple Sclerosis/prevention & controlABSTRACT
Human brucellosis exhibits diverse pathological manifestations that can affect almost any organ. In particular, osteoarticular complications are the most common focal manifestation of brucellosis and occur in 40-80% of patients. In immunocompetent mice, Brucella replication is generally restricted to the spleen, liver, and to a lesser extent, LNs, thereby limiting their use for study of focal inflammation often found in brucellosis. Here, we report that nasal, oral, or peritoneal infection of IFN-γ(-/-) mice with WT Brucella melitensis or Brucella abortus results in joint and periarticular tissue inflammation. Histological analysis of the affected joints revealed inflammatory infiltrates and debris within the joint space colocalizing with Brucella antigen. Osteoarthritis, necrosis, periarticular soft tissue inflammation, and substantial brucellae burdens were observed. Oral rifampicin was effective in clearing infection and halting further progression of focal inflammation from infected IFN-γ(-/-) mice, although some symptoms and swelling remained. Elevated IL-1 ß, but not TNF-α, IL-6, or IL-17, was detected in joint homogenates from infected IFN-γ(-/-) mice. Whereas more susceptible to systemic infection, IL-1R(-/-) mice depleted of IFN-γ were more resistant to focal inflammation than WT mice similarly depleted of IFN-γ. Collectively, these results show IFN-γ(-/-) mice represent a potential model for study of focal inflammation attributed to Brucella infection and will allow evaluation of intervention strategies targeting IL-1, IL-1R, or other inflammatory mediators, with the potential to complement antibiotic-based therapies.
Subject(s)
Brucellosis/immunology , Dermatitis/immunology , Inflammation Mediators/physiology , Interferon-gamma/deficiency , Interleukin-1/physiology , Myositis/immunology , Animals , Brucellosis/genetics , Brucellosis/microbiology , Cells, Cultured , Dermatitis/genetics , Dermatitis/microbiology , Disease Models, Animal , Humans , Inflammation/immunology , Inflammation/microbiology , Inflammation/pathology , Interferon-gamma/physiology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Myositis/genetics , Myositis/microbiology , RabbitsABSTRACT
Human IBD, including UC and Crohn's disease, is characterized by a chronic, relapsing, and remitting condition that exhibits various features of immunological inflammation and affects at least one/1000 people in Western countries. Polyphenol extracts from a variety of plants have been shown to have immunomodulatory and anti-inflammatory effects. In this study, treatment with APP was investigated to ameliorate chemically induced colitis. Oral but not peritoneal administration of APP during colitis induction significantly protected C57BL/6 mice against disease, as evidenced by the lack of weight loss, colonic inflammation, and shortening of the colon. APP administration dampened the mRNA expression of IL-1ß, TNF-α, IL-6, IL-17, IL-22, CXCL9, CXCL10, CXCL11, and IFN-γ in the colons of mice with colitis. APP-mediated protection requires T cells, as protection was abated in Rag-1(-/-) or TCRα(-/-) mice but not in IL-10(-/-), IRF-1(-/-), µMT, or TCRδ(-/-) mice. Administration of APP during colitis to TCRα(-/-) mice actually enhanced proinflammatory cytokine expression, further demonstrating a requirement for TCRαß cells in APP-mediated protection. APP treatment also inhibited CXCR3 expression by TCRαß cells, but not B or NK cells, in the colons of mice with colitis; however, depletion of CD4(+) or CD8(+) T cells alone did not abolish APP-mediated protection. Collectively, these results show that oral administration of APP protects against experimental colitis and diminishes proinflammatory cytokine expression via T cells.
Subject(s)
Colitis/immunology , Cytokines/antagonists & inhibitors , Dextran Sulfate/toxicity , Inflammation Mediators/physiology , Malus/physiology , Polyphenols/physiology , T-Lymphocyte Subsets/drug effects , T-Lymphocyte Subsets/pathology , Animals , Cells, Cultured , Colitis/chemically induced , Colitis/pathology , Cytokines/biosynthesis , Cytokines/genetics , Dextran Sulfate/administration & dosage , Female , Humans , Inflammation Mediators/antagonists & inhibitors , Inflammation Mediators/chemistry , Male , Malus/chemistry , Mice , Mice, Inbred C57BL , Mice, Knockout , Polyphenols/chemistry , T-Lymphocyte Subsets/immunologyABSTRACT
Receptor-mediated gene transfer using an M cell ligand has been shown to be an efficient method for mucosal DNA immunization. To investigate further into alternative M cell ligands, the plant lectin, Ulex europaeus agglutinin I (UEA-1), was tested. UEA-1 binds to human intestinal Caco-2 cells, and these cells can be transfected with poly-l-lysine (PL)-conjugated UEA-1 for expression of reporter cDNAs. When tested in vivo, mice nasally immunized with UEA-1-PL complexed to plasmid encoding HIV-1 envelope showed elevated systemic and mucosal antibody responses, and these were supported by tissue antibody-forming cells. Likewise, elevated envelope-specific CTLs were induced. Thus, UEA-1 mediated DNA delivery represents an alternative mucosal formulation for inducing humoral and cellular immunity against HIV-1.