Unexpected T cell regulatory activity of anti-histone H1 autoantibody: its mode of action in regulatory T cell-dependent and -independent manners.

Induction of anti-nuclear antibodies against DNA or histones is a hallmark of autoimmune disorders, but their actual contribution to disease predisposition remains to be clarified. We have previously reported that autoantibodies against histone H1 work as a critical graft survival factor in a rat model of tolerogeneic liver transplantation. Here we show that an immunosuppressive anti-histone H1 monoclonal antibody (anti-H1 mAb) acts directly on T cells to inhibit their activation in response to T cell receptor (TCR) ligation. Intriguingly, the T cell activation inhibitory activity of anti-H1 mAb under suboptimal dosages required regulatory T (Treg) cells, while high dose stimulation with anti-H1 mAb triggered a Treg cell-independent, direct negative regulation of T cell activation upon TCR cross-linking. In the Treg cell-dependent mode of immunosuppressive action, anti-H1 mAb did not induce the expansion of CD4(+-)Foxp3(+) Treg cells, but rather potentiated their regulatory capacity. These results reveal a previously unappreciated T cell regulatory role of anti-H1 autoantibody, whose overproduction is generally thought to be pathogenic in the autoimmune settings.

A novel peptide mimotope identified as a potential immunosuppressive vaccine for organ transplantation.

We reported that anti-histone H1 autoantibody is one of the main immunosuppressive factors in serum that is induced after orthotopic liver transplantation in a rat tolerogenic model. We generated a novel anti-histone H1 IgM mAb produced by hybridoma 16G9 (16G9 mAb) that shows MLR-inhibitory activity. Identification of a functional epitope responsible for the immunosuppressive activity of 16G9 mAb may lead to the establishment of a novel therapeutic strategy. We used a combinatorial phage display peptide library to screen for peptides that bind to 16G9 mAb. Consequently, two peptides that bind to 16G9 mAb, SSV and LPQ, were selected from the library. The binding of 16G9 mAb to histone H1 was inhibited by SSV. SSV was recognized by rat tolerogenic post-orthotopic liver transplantation serum and the binding to SSV was inhibited by histone H1. Mice were immunized with keyhole limpet hemocyanin-conjugated SSV and LPQ. Abs induced by SSV immunization inhibited Con A-stimulated splenocyte proliferation, and the inhibition was neutralized by preincubation with SSV. Splenocytes stimulated by anti-CD3 Ab were inhibited by SSV-induced Abs using CFSE labeling. SSV immunization in rats before heterotopic heart transplantation resulted in significant prolonged allograft survival. These findings suggested that SSV is a functional histone H1-binding epitope for 16G9 mAb. SSV is capable of determining serum immunoreactivity against histone H1 as an index marker for tolerance. The inhibitory activity of SSV-induced Abs on blast cell proliferation and the prolonged graft survival that results from SSV immunization imply a potential for the development of an immunosuppressive vaccine.

Sunlight Exposure and Phototherapy: Perspectives for Healthy Aging in an Era of COVID-19.

Most humans depend on sunlight exposure to satisfy their requirements for vitamin D3. However, the destruction of the ozone layer in the past few decades has increased the risk of skin aging and wrinkling caused by excessive exposure to ultraviolet (UV) radiation, which may also promote the risk of skin cancer development. The promotion of public health recommendations to avoid sunlight exposure would reduce the risk of skin cancer, but it would also enhance the risk of vitamin D3 insufficiency/deficiency, which may cause disease development and progression. In addition, the ongoing global COVID-19 pandemic may further reduce sunlight exposure due to stay-at-home policies, resulting in difficulty in active and healthy aging. In this review article, we performed a literature search in PubMed and provided an overview of basic and clinical data regarding the impact of sunlight exposure and vitamin D3 on public health. We also discuss the potential mechanisms and clinical value of phototherapy with a full-spectrum light (notably blue, red, and near-infrared light) as an alternative to sunlight exposure, which may contribute to combating COVID-19 and promoting active and healthy aging in current aged/superaged societies.

Immunological aspects and therapeutic significance of an autoantibody against histone H1 in a rat model of concanavalin A-induced hepatitis.

We previously demonstrated the immunosuppressive activity of anti-histone H1 autoantibody induced in experimental and clinical liver allograft tolerance. This study aimed to explore the immunological aspects of anti-histone H1 autoantibody in liver injury induced by concanavalin A (Con A). To establish a Con A-hepatitis model, 20 mg/kg Con A was intravenously injected into rats, after which liver function and histopathological analyses were performed. In this model, anti-histone H1 autoantibody was transiently induced in the sera during the natural recovery stage, 3-7 days after Con A injection. To evaluate the therapeutic significance of anti-histone H1 autoantibody, a polyclonal antibody against histone H1 was intraperitoneally injected immediately after Con A injection. We found that injection of anti-histone H1 antibody could reduce Con A-induced liver damage. Further mechanical analyses revealed that anti-histone H1 antibody altered the intracellular activation of mitogen-activated protein kinase, nuclear factor-kappaB and calcineurin via T-cell receptor signalling, suggesting that anti-histone H1 antibody may protect the liver from Con A-induced injury by inhibiting activation of effector T cells. These findings suggest that anti-histone H1 autoantibody may be a natural immune regulatory factor that protects inflamed livers suffering from autoimmune hepatitis and may lead to T-cell unresponsiveness through the selective regulation of mitogen-activated protein kinase/nuclear factor-kappaB and calcineurin signalling.

Involvement of autoimmunity against nuclear histone H1 in liver transplantation tolerance.

BACKGROUND: Our recent studies suggested that anti-histone H1 autoantibody (auto-Ab) plays an important role in experimental and clinical liver allograft tolerance as a natural immunosuppressive factor. The present study aimed to explore how the autoimmune response against histone H1 is involved in tolerance induction. METHODS: The measurement of anti-histone H1 auto-Ab and immunohistochemical analysis were performed in serum and liver allografts after orthotopic liver transplantation (OLT). To compare the auto-Ab response against histone H1 between the recipients of rejector (DA-LEW) and tolerogenic (DA-PVG) OLT models, naïve recipients were immunized with calf thymus histone H1. The immunosuppressive state of histone H1-immunized rats was assayed by mixed lymphocyte reaction (MLR). RESULTS: Anti-histone H1 Ab titer was transiently increased during the rejection phase after OLT (days 7-21) in the DA-PVG combination, while no such response was confirmed in the DA-LEW acute rejection model. Nuclear histone H1 antigens were found in the cytoplasm and the extracellular environment in liver allografts at the rejection phase in the tolerogenic model but not in the rejector model, resulting from the transient induction of anti-histone H1 auto-Ab in recipient PVG rats after OLT. Low dose and short-term immunization with histone H1 upregulated the anti-histone H1 Ab titer in naïve PVG rats, which exhibited a low allogeneic immune response, while no such response was found in naïve LEW rats. CONCLUSIONS: These results suggest that the sensitivity to nuclear antigens such as histone H1 may be a key factor determining the acceptance or rejection of donor liver grafts, at least in DA-PVG and DA-LEW combinations.

Experimental and clinical significance of antinuclear antibodies in liver transplantation.

Histone H1 and high-mobility group box 1 (HMGB1) proteins are known to initiate an immune reaction, and the corresponding antibodies (Abs) possess immunosuppressive activity. In the present study, we aimed to evaluate the immunological role of antinuclear Abs in experimental and clinical liver transplantation. In a rat tolerogenic orthotopic liver transplantation (OLT) model, antihistone H1 and HMGB1 titers were induced during the rejection and tolerance induction phases, respectively. Those Ab responses also were confirmed in a drug-induced tolerance model (acute rejection model + cyclosporin A [0 to 14 days after OLT]). We also found a similar tendency in our clinical drug-free patient (who experienced complete cessation of any immunosuppressive treatments) and that antinuclear Abs induced in the serum after cessation of immunosuppressants play a part of immune privilege in this patient. These results suggest that antinuclear Abs are important factors for overcoming rejection and the subsequent tolerance induction in liver transplantation.

The immunological role of lipid transfer/metabolic proteins in liver transplantation tolerance.

BACKGROUND: In a rat tolerogenic orthotopic liver transplantation (OLT) model, recipient serum after OLT (post-OLT serum) has been reported to prevent allograft rejection. A previous proteomic study indicated that apolipoprotein E (apo-E), which is an important factor for cholesterol transportation, is expressed at the latter tolerogenic phase after OLT. It has also been known that adipose tissue-derived adipokine, adiponectin, is an essential factor for fatty acid catabolism. This study aimed to characterize the role of lipid transfer/metabolic proteins in liver transplantation tolerance. METHODS: To identify the apo-E and adiponectin in post-OLT serum, Western analyses and enzyme-linked immunosorbent assay (ELISA) were performed, respectively. The immunosuppressive activities of those factors were evaluated by inhibition of the mixed lymphocyte reaction (MLR). RESULTS: Western analyses showed that the mobility of apo-E was shifted at the latter tolerogenic phase after OLT in a natural tolerance model, and a similar phenomenon was confirmed in the serum of a drug-induced tolerance model (rejection model+cyclosporin A (CsA); 0 to 14 days) after cessation of CsA. Further study revealed that neutralization of modified apo-E in post-OLT serum reduced the immunosuppressive activity. Additionally, plasma adiponectin was significantly elevated at the latter phase after OLT, and possessed MLR-inhibitory activity. CONCLUSIONS: These results suggest that the mobility shift of apo-E and/or the up-regulation of adiponectin may be necessary for overcoming the rejection, recovering the liver allograft function, and following tolerance induction in experimental OLT models, and may be useful as one indicator to surmise the prognosis after liver transplantation.

Impact of vaccine therapy using nuclear histone H1 on allograft survival in experimental organ transplantation.

BACKGROUND: We recently reported that autoreactive antibody (Ab) against nuclear histone H1 had been identified as an immunosuppressive factor in a rat tolerogenic orthotopic liver transplantation (OLT) model. The present study aimed to determine whether the up-regulation of antihistone H1 Ab by histone H1 vaccination leads to tolerance. METHODS: Histone H1-immunized rats were established by intraperitoneal vaccination with histone H1 at every two-weekly interval. By using mixed lymphocyte reaction (MLR) and heterotopic heart transplantation (HHT), the alloreactive T cell response and allograft survival of histone H1-immunized rats were compared with those of control rats. Cytokine and cellular profiles in histone H1-immunized rats were determined by reverse transcriptase polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay (ELISA) and flow cytometry. RESULTS: Immunization with histone H1 in Freund's adjuvant induced alloreactive T cell unresponsiveness and prolonged heterotopic heart allograft survival. It also down-regulated the expression of major histocompatibility complex (MHC) class II and CD25 on splenic cells, elevated the T helper cell type 2 (Th2) skewing index (Interleukin (IL)-4/interferon (IFN)-gamma ratio or IL-4/IL-2 ratio) and modified the serum cytokine profiles. CONCLUSIONS: The present results suggest that histone H1 vaccination of transplant recipients, which leads to the production of immunosuppressive factor and the modification of the cytokine/cellular profiles, has great potential as a tolerance therapy for prospective transplantation.

Skin permeability of tulobuterol in two transdermal formulations and their followability.

Various generic transdermal formulations of tulobuterol containing rubber and acrylate base polymers are commercially available in Japan. However, none of the formulations have been compared directly with respect to the skin permeability of tulobuterol and to their follow ability. Tulobuterol Tape Sawai of rubber base and Tulobuterol Tape NP of acrylate base were used to conduct the in vitro 24-hour skin permeability test of tulobuterol at receiver solution temperatures of 32°C, 37°C, and 40°C. Furthermore, the followability of these tapes were examined by measuring the depth of the pores that were formed in their adhesive layer. Consequently, the maximum flux of tulobuterol was greater for Tulobuterol Tape NP. Arrhenius plot analysis revealed that Tulobuterol Tape Sawai was more sensitive to skin surface temperature compared with Tulobuterol Tape NP. Skin abrasion had a greater effect on the skin permeability of tulobuterol in Tulobuterol Tape Sawai than in Tulobuterol Tape NP. Followability was greater for Tulobuterol Tape NP than for Tulobuterol Tape Sawai. These results suggest that a transdermal formulation of acrylate base is preferable to that with a rubber base when skin surface temperature varies or when the skin is abraded. In clinical settings, therefore, a formulation of acrylate base is preferable to a formulation of rubber base when skin surface temperature varies or when the skin is abraded. The formulation needs to be applied to the skin of less asperity for the achievement of better transdermal absorption of tulobuterol.

Impact of Histone H1 on the Progression of Allergic Rhinitis and Its Suppression by Neutralizing Antibody in Mice.

Nuclear antigens are known to trigger off innate and adaptive immune responses. Recent studies have found that the complex of nucleic acids and core histones that are derived from damaged cells may regulate allergic responses. However, no fundamental study has been performed concerning the role of linker histone H1 in mast cell-mediated type I hyperreactivity. In this study, we explored the impact of histone H1 on mast cell-mediated allergic responses both in vitro and in vivo. In the course of a bona-fide experimental allergen sensitization model upon co-injection with alum adjuvant, ovalbumin (OVA), but not PBS, induced elevated levels of circulating histone H1. Intranasal challenge with histone H1 to OVA/alum- (but not PBS/alum)-sensitized mice induced significantly severer symptoms of allergic rhinitis than those in mice sensitized and challenged with OVA. A monoclonal antibody against histone H1 not only suppressed mast cell degranulation, but also ameliorated OVA-induced nasal hyperreactivity and IgE-mediated passive cutaneous anaphylaxis. Our present data suggest that nuclear histone H1 represents an alarmin-like endogenous mediator acting on mast cells, and that its blockage has a therapeutic potential for mast cell-mediated type I hyperreactivity.

A novel anti-histone H1 monoclonal antibody, SSV monoclonal antibody, improves lung injury and survival in a mouse model of lipopolysaccharide-induced sepsis-like syndrome.

BACKGROUND: Histones play important roles in both host defenses and inflammation related to microbial infection. A peptide mimotope (SSV) was identified from a novel histone H1 monoclonal antibody (16G9 mAb) that was shown to inhibit the mixed lymphocyte reaction. In the present study, an anti-SSV producing hybridoma was established. We investigated the effects of SSV mAb in a mouse acute inflammation model induced by intraperitoneal injection of lipopolysaccharide (LPS). METHODS: SSV mAb was generated and characterized. Mice were treated with SSV mAb or a control IgG antibody prior to LPS injection. Evaluation of survival rate and lung tissue on histological score was performed. The levels of inflammatory cytokines and histones H1, H3, and H4 in plasma and lung tissue were measured by ELISA. RESULTS: Competitive ELISA revealed that SSV mAb binds to histone H1. SSV mAb improved lung injury and prolonged the survival of LPS-injected mice. Increased levels of histones H1, H3, and H4 and inflammatory cytokines (TNF-α, IL-1ß, and IL-6) in plasma and lung tissue after LPS injection were ameliorated by SSV mAb. CONCLUSION: SSV mAb is shown to have anti-inflammatory activity and organ-protective effects, highlighting the importance of controlling histone H1 as well as H3 and H4 levels during inflammation.

The search for immunosuppressive therapies to induce tolerance in organ transplantation.

Organ transplantation has become a major therapeutic option for patients with irreversible organ diseases. Immunosuppressive agents are usually required to prevent allograft rejection in patients who undergo an organ transplantation. Such drugs suppress both specific and nonspecific immunity, and render the recipient more susceptible to both infection and malignancy. Therefore, the development of more effective and less toxic immunosuppressive agents could improve the clinical outcomes of organ transplant recipients. Since the early days of clinical and experimental liver transplantation, it has been known that the liver is less likely to be rejected in comparison to other organs and may be tolerogenic even across a fully allogeneic MHC barrier in some specific cases. Spontaneous acceptance of liver allografts has been observed in several species. Orthotopic liver transplantation (OLT) in certain rat strains is accepted without immunosuppressive agents and serum from post-OLT recipients displays immunosuppressive activity. Attempts have been made to identify the immunosuppressive factors that are present in post-OLT serum to elucidate the mechanism of immunological tolerance and to discover novel immunosuppressive agents for potential use in organ transplantation. In this review we will focus on established and recent findings in the identification of immunosuppressive factors in a rat tolerogenic OLT model. The most recent therapeutic methods in organ transplantation and future prospects will be discussed.

Development of a two-step chromatography procedure that allows the purification of a high-purity anti-histone H1 monoclonal immunoglobulin M antibody with immunosuppressant activity.

In organ transplantation, the development of a novel immunosuppressant free of the need for permanent administration and any serious side effects has eagerly been awaited. We have previously reported that an anti-histone H1 polyclonal antibody has immunosuppressant activity. Here we prepared an anti-histone H1 monoclonal antibody as an analytical tool to elucidate its mechanism of immunosuppression. The isotype of this monoclonal antibody was immunoglobulin M. A monoclonal antibody prepared for administration to organ transplantation model animals should not contain any allogenic proteins and should have high purity. Therefore, we conducted a two-step chromatography procedure, consisting of strong anion-exchange chromatography and gel filtration chromatography, to purify an anti-histone H1 monoclonal immunoglobulin M antibody from the serum-free culture supernatant of hybridomas. Consequently, we successfully purified the monoclonal antibody at 96%, a purification rate at which its administration to organ transplantation model animals is possible.

Dendritic cell immunotherapy with poly(D,L-2,4-diaminobutyric acid)-mediated intratumoral delivery of the interleukin-12 gene suppresses tumor growth significantly.

A conventional DC-based immunotherapy has been tested clinically for treatment of patients with advanced cancer but requires modification to further improve the clinical results. In this study, we evaluated the in vivo antitumor effects of DC therapy, non-viral-mediated IL-12 gene therapy, and a combination of the two in a murine bilateral subcutaneous tumor model. DC therapy alone and IL-12 gene therapy alone suppressed tumor growth at the injected sites. However, the antitumor effect on the distant contralateral tumor was insufficient. When DC therapy and IL-12 gene therapy were carried out simultaneously, tumor growth was significantly suppressed bilaterally (P < 0.001). Cytolytic activity was augmented significantly in mice given the combination treatment compared to in mice treated with either DC or IL-12 gene therapy alone (P < 0.05). Microvessel density of both tumors was significantly lower in mice subjected to the combination therapy than in mice treated otherwise (P < 0.05). Furthermore, no side-effects were observed in the treated mice. DC therapy combined with non-viral-mediated intratumoral IL-12 gene delivery has a synergistic antitumor effect not only on targeted tumors but also on contralateral distant tumors and may be of great potential as a therapeutic treatment for patients with advanced cancer.