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Dominant-negative TGF-β receptor II (dnTGF-βRII) mice spontaneously develop an autoimmune cholangitis resembling human primary biliary cirrhosis (PBC). Interestingly, the dominant-negative TGF-β receptor is expressed by both CD4(+) and CD8(+) T cells and leads to greatly reduced (but not absent) TGF-β signaling resulting in T cell intrinsic cell mediated autoimmunity. However, the mechanisms of the T cell dysregulation remain unclear. Recently it has been shown that TGF-β signaling is intimately involved with miRNA biogenesis and control. Herein we show that lack of T cell TGF-β signaling leads to down regulation of T cell miRNAs but up-regulation of the key inflammatory miRNA 21. Furthermore, the expression of miR-21 from hepatic effector CD8(+) T cells is significantly higher than in the same subsets isolated from spleen and mesenteric lymph nodes of the dnTGF-βRII mice. Previous studies indicate that miR-21 increases the synthesis of IFN-γ and IL-17A by T cells and suppresses apoptosis via programmed cell death protein 4 (PDCD4). Data presented herein demonstrate that transfecting w.t. B6 T cell subsets with miR-21 resulted in up-regulation of the inflammatory cytokines TNF-α and IFN-γ, thus partly replicating the dnTGF-βRII T cell phenotype. In conclusion, these data suggest miR-21 plays a critical role in the production of pro-inflammatory cytokines in dnTGF-βRII mice, which could be a contributing factor for the development of the organ-specific autoimmune cholangitis and colitis in this murine model of human PBC., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In an attempt to simplify the protocol of DC generation in vitro, studies conducted herein show that functional DCs could be generated from bulk peripheral blood mononuclear cells (PBMCs) in media containing GM-CSF and IL-4. Interestingly, when PBMCs, but not purified monocytes, were exposed to either CCR5- or CXCR4-tropic inactivated HIV-1 isolates (iHIV-1) at the initiation of the culture, DC yields were significantly reduced in a dose-dependent manner because of monocyte apoptosis. Similar impairment of DC generation was noted using type I IFNs and poly IC not only in cultures of PBMCs but also using highly enriched monocytes. This effect was reversed by antihuman type I IFN receptor, but not by anti-FasL, anti-TRAIL, anti-TNF, or a mixture of these antibodies. iHIV-1-exposed PBMCs, but not monocytes, produced high levels of IFN-alpha but not IFN-beta. PBMCs depleted of CD123(+) plasmacytoid DCs produced low levels of IFN-alpha and were resistant to iHIV-1-mediated DC impairment. Interestingly, exogenously added TNF reversed the impairment by iHIV-1 in the PBMC cultures. In conclusion, the present results indicate that iHIV-1 impairs the in vitro generation of functional DCs from PBMCs through the induction of IFN-alpha from plasmacytoid DCs in a CD4-dependent fashion in the absence of TNF., (Copyright 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Recently, we identified a child born with a genetic deficiency of IL-2 receptor alpha (IL-2Ralpha, CD25) expression who had several clinical manifestations of primary biliary cirrhosis (PBC). In addition, there has been suggestive evidence in both patients with PBC and their first-degree relatives that a deficiency of regulatory T cells (Tregs) is an integral component for susceptibility to PBC. Based on these observations, we generated IL-2Ralpha/CD25 deficient (IL-2Ralpha(-/-)) mice and wild-type littermate controls and followed them longitudinally for the natural history of liver immunopathology and appearance of antimitochondrial antibodies (AMAs). The analyses included immunohistochemical staining of liver and portal tract infiltrates as well as FACS profiles of lymphoid subpopulations in liver and spleen. In addition, serum cytokine profiles were quantitated. Importantly, IL-2Ralpha(-/-), but not littermate controls, develop portal inflammation and biliary ductular damage similar to human patients with PBC. CD4(+) and CD8(+) T cells predominate among portal cell infiltrates and sera reflect a Th1 cytokine bias with increased levels of IFN-gamma, TNF-alpha, IL-2 and IL-12p40. Of importance is the finding that the IL-2Ralpha(-/-) mice not only develop significantly increased serum levels of IgG and IgA, but they also develop AMAs with specificity for PDC-E2, which maps to the inner lipoyl domain of the autoantigen, all characteristics which are hallmarks of human PBC. In conclusion, the IL-2Ralpha(-/-) mice should facilitate studies of the early events in PBC and especially the tantalizing connection between Treg deficiency and autoimmunity specifically directed to mitochondrially located PDC-E2 and subsequent biliary ductular cell damage.

Although the pathogenesis of primary biliary cirrhosis (PBC) remains enigmatic, the immune system plays a key role in the initiation and subsequent development of pathology. Previous studies have indicated a critical role of the innate immune system. Importantly, natural killer (NK) cells are abundant in liver where they serve as sentinels of the immune system. In addition, NK cells have significant biologic activity based on their production of immunoregulatory cytokines. To address this issue, we have investigated several qualitative and quantitative activities of NK cells in patients with PBC as well as normal and liver diseased controls. We report herein a marked increase in the frequency and absolute number of blood and liver NK cells in PBC patients. Moreover, the cytotoxic activity and perforin expression by isolated NK cells were significantly increased in PBC patients associated with increased levels of plasma IL-8 and the expression of CD128a (IL-8 receptor) on NK cells. In contrast, the levels of IFN-gamma, IL-6 and IL-8 synthesized by NK cells were significantly decreased in PBC patients as compared to controls. In conclusion, data from this study provide compelling evidence supporting a biologic role of NK cells in the immunopathogenesis of PBC.

The shift from a predominant synthesis of prototype Th1 cytokines to Th2 or Th0 type of cytokines by antigen activated PBMC's from HIV infected humans and SIV infected disease susceptible rhesus macaques (RM) has been shown to be associated with disease progression. Paradoxically, antigen activated PBMC's from sooty mangabeys (SM), which are naturally infected with SIV and are disease resistant despite high viral loads, maintain a predominant Th2 cytokine profile. It has been reasoned that the resistance to perturbations of cytokine synthesis by slow and/or nonprogressor HIV infected patients and SIV infected disease susceptible RM is secondary to inherited polymorphisms within the promoter regions for cytokines. Similar promoter polymorphisms could also contribute to the cytokine profile of PBMC's from SM. To address this issue promoter regions for the major Th1/Th2 cytokines from RM and SM were cloned and sequenced. Sequence analysis of promoter fragments of IL-4, IL-10, IL-12 p40, IFN-gamma and TNF-alpha from the two monkey species showed varying degree of homology ranging from high degree of homology detected for IFN-gamma promoter (> 99%) to relatively high degree of polymorphism detected for TNF-alpha promoter (94% homology). In addition, several variable regions within the promoters of IL-12 p40, IL-10 and TNF-alpha in the two species contain polymorphisms in sequences that constitute binding sites of known transcription factors (TF). Such differences are likely to differentially bind TF and thus either qualitatively and/or quantitatively affect the regulation of cytokine synthesis in these two species and potentially contribute to disease progression and/or resistance.

Outbreaks of infectious diseases such as HIV and the much televised and attention-getting outbreaks of diseases such as Ebola, Hantaviruses, and the most recent outbreak of SARS have induced a significant new interest in the formulations and more importantly the science of vaccinology, which has previously to a large extent been conducted empirically. Our laboratory has focused on the use of recombinant nonhuman primate cytokines as adjunctive therapies for inducing antigen-specific immune responses in monkeys because most recombinant human cytokines appear to be immunogenic. This article provides a summary of our work with such cytokines, which includes attempts to define optimum dosing schedules that lead to optimal primary and lasting memory antigen-specific immune responses.

The Coronavirus-associated disease, that was first identified in 2019 in China (CoViD-19), is a pandemic caused by a bat-derived beta-coronavirus, named SARS-CoV2. It shares homology with SARS and MERS-CoV, responsible for past outbreaks in China and in Middle East. SARS-CoV2 spread from China where the first infections were described in December 2019 and is responsible for the respiratory symptoms that can lead to acute respiratory distress syndrome. A cytokine storm has been shown in patients who develop fatal complications, as observed in past coronavirus infections. The management includes ventilatory support and broad-spectrum antiviral drugs, empirically utilized, as a targeted therapy and vaccines have not been developed. Based upon our limited knowledge on the pathogenesis of CoViD-19, a potential role of some anti-rheumatic drugs may be hypothesized, acting as direct antivirals or targeting host immune response. Antimalarial drugs, commonly used in rheumatology, may alter the lysosomal proteases that mediates the viral entry into the cell and have demonstrated efficacy in improving the infection. Anti-IL-1 and anti-IL-6 may interfere with the cytokine storm in severe cases and use of tocilizumab has shown good outcomes in a small cohort. Baricitinib has both antiviral and anti-inflammatory properties. Checkpoints inhibitors such as anti-CD200 and anti-PD1 could have a role in the treatment of CoViD-19. Rheumatic disease patients taking immunosuppressive drugs should be recommended to maintain the chronic therapy, prevent infection by avoiding social contacts and pausing immunosuppressants in case of infection. National and international registries are being created to collect data on rheumatic patients with CoViD-19.

Primary biliary cirrhosis (PBC) is characterized by an intense biliary inflammatory CD4(+) and CD8(+) T cell response. Very limited information on autoantigen-specific cytotoxic T lymphocyte (CTL) responses is available compared with autoreactive CD4(+) T cell responses. Using peripheral blood mononuclear cells (PBMCs) from PBC, we identified an HLA-A2-restricted CTL epitope of the E2 component of pyruvate dehydrogenase (PDC-E2), the immunodominant mitochondrial autoantigen. This peptide, amino acids 159-167 of PDC-E2, induces specific MHC class I-restricted CD8(+) CTL lines from 10/12 HLA-A2(+) PBC patients, but not controls, after in vitro stimulation with antigen-pulsed dendritic cells (DCs). PDC-E2-specific CTLs could also be generated by pulsing DCs with full-length recombinant PDC-E2 protein. Furthermore, using soluble PDC-E2 complexed with either PDC-E2-specific human monoclonal antibody or affinity-purified autoantibodies against PDC-E2, the generation of PDC-E2-specific CTLs, occurred at 100-fold and 10-fold less concentration, respectively, compared with soluble antigen alone. Collectively, these data demonstrate that autoantibody, helper, and CTL epitopes all contain a shared peptide sequence. The finding that autoantigen-immune complexes can not only cross-present but also that presentation of the autoantigen is of a higher relative efficiency, for the first time defines a unique role for autoantibodies in the pathogenesis of an autoimmune disease.

Background Pertussis toxin ( PTX) blocks GPCR signaling resulting in the inhibition of chemotaxis/cell adhesion. It was reasoned that inhibition of cell trafficking may be an approach to prevent HIV/ SIV transmission. Methods In this study, PTX in HEC gel was applied to the vaginal wall of monkeys that were then challenged intravaginally with SIVmac251. Results Results of these studies showed that 2 of 4 animals were resistant to infection. Furthermore, infection was correlated with a marked increase in the plasma and cervicovaginal lavage levels of select chemokines and cytokines. Conclusions Results from this preliminary feasibility study dictate that further studies that include a larger number of animals are required to optimize this protocol and establish the efficacy of this approach. In addition, such future studies will provide important information on the role of specific chemokines that play a role in lymphocyte trafficking within the genital tract and serve as additional therapeutic targets. [ABSTRACT FROM AUTHOR]

Background OX40 ligand (OX40L) co-stimulates and differentiates T cells via ligation of OX40 that is transiently induced on T cells upon activation, resulting in prolonged T cell survival and enhanced cytokine production by T cells. This view has led to the targeting of OX40 as a strategy to boost antigen specific T cells in the context of vaccination. In addition, the ligation of OX40 has also been shown to inhibit infection by CCR5-utilizing (R5) but not CXCR4-utilizing (X4) human immunodeficiency virus type-1 (HIV-1) via enhancement of production of CCR5-binding β-chemokines. It was reasoned that human T cell leukemia virus type-I (HTLV-1) immortalized T cell lines that express high levels of OX40L could serve as an unique source of physiologically functional OX40L. The fact that HTLV-1+ T cell lines simultaneously also express high levels of OX40 suggested a potential limitation. Results Results of our studies showed that HTLV-1+ T cell lines bound exogenous OX40 but not OX40L, indicating that HTLV-1+ T cell lines express an active form of OX40L but an inactive form of OX40. Anti-OX40 non-blocking monoclonal antibody (mAb), but not blocking mAb, stained HTLV-1+ T cell lines, suggesting that the OX40 might be saturated with endogenous OX40L. Functionality of the OX40L was confirmed by the fact that a paraformaldehyde (PFA)-fixed HTLV-1+ T cell lines inhibited the infection of autologous activated peripheral blood mononuclear cells (PBMCs) with R5 HIV-1 which was reversed by either anti-OX40L blocking mAb or a mixture of neutralizing mAbs against CCR5- binding β-chemokines. Conclusions Altogether, these results demonstrated that autologous T cell lines immortalized by HTLV-1 can be utilized as a conventional source of physiologically functional OX40L. [ABSTRACT FROM AUTHOR]

Compares the serum cytokine levels of patients who died of Ebola hemorrhagic fever (EHF) with those of patients who recovered from the disease. Marked elevation of interferon-gamma levels observed in sequential serum samples from all fatal EHF cases; Factors that contribute to a fatal outcome in EHF patients.

Isolated intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) from jejunum of SIV infected animals were examined for alterations in basal cytokine expression by RT-PCR. Remarkable changes in IFNγ and IL-10 RNA levels were observed in IEL and LPL in SIV infection while IL-4 and IL-2 RNA levels remained unaltered In addition, the CD4+ and CD8+ LPL were examined for intracellular cytokine production following mit0genic activation by flowcytometry, Both CD4+ and CD8+ T lymphocytes in intestinal mucosa retained the potential to produce IFNγ in response to mitogenic stimulation in vitro, without a remarkable change in IL-4 production. The dominant IFNγ cytokine response could be one of the major contributing factors in SIV associated enteropathy. [ABSTRACT FROM AUTHOR]

Several human CC chemokines have been shown to inhibit HIV/ SIV infection in vitro, providing the rationale for their potential use in vivo. However, because of their inherent physiological effect, such chemokines are reasoned to be of limited therapeutic value due to potential side effects. The knowledge that amino terminus modified Or deleted human RANTES retains its receptor binding properties but loses its signaling properties has provided a means to use such modified chemokines in vivo for possible therapeutic benefits. In efforts to test the efficacy of such modified chemokines, our laboratory has cloned, sequenced, and prepared recombinant forms Of wild-type (wt) and amino-terminus modified rhesus macaque chemokines MIP-1α, MIP1β, and RANTES. These Sets of Chemokines were tested for their potential to inhibit SIV infection and induce signaling. The data showed that whereas wt chemokines retained both virus inhibitory and signaling functions, corresponding amino-terminus modified chemokines only showed vitals inhibitory effects without detectable signaling effects. Such reagents will be valuable for evaluation of their therapeutic potential in vivo, either alone or as adjuncts to other chemotherapeutic drugs. [ABSTRACT FROM AUTHOR]

IL-21 regulates Th17 cell homeostasis, enhances the differentiation of memory B cells and antibody-secreting plasma cells, and promotes the maintenance of CD8 + T-cell responses. In this study, we investigated the phenotype, function, and frequency of blood and Intestinal IL-21-- producing cells in nonhuman primates that are hosts of progressive (rhesus macaques [RMs]) and nonprogressive (sooty mangabeys [SMs]) SIV infection. We found that, in both species, memory Introduction CD4+CD95+CCR6" T cells are the main IL-21 producers, and that only a small fraction of CD4+IL-21+ T cells produce IL-17. During chronic SIV infection of RMs, CD4+IL-21+ T cells were significantly de-pleted in both blood and rectal mucosa, with the extent of this depletion correlat-ing with the loss of Th17 cells. Further-more, treatment with IL-21 increased the in vivo levels of Th17 cells in SIV-infected RMs. In contrast, normal levels of CD4+IL-21+ T cells were found in SIV-infected SMs. Collectively, these data indicate that depletion of IL-21-producing CD4+ T cells distinguishes progressive from nonpro-gressive SIV Infection of RMs and SMs, and suggest that depletion of CD4+IL-21+ T cells Is involved in the preferential loss of Th17 cells that is associated with SIV disease progression. Further preclinical studies of IL-21 as a potential immuno-therapeutlc agent for HIV infection may be warranted. (Blood. 2012;120(19): 3925-3935) [ABSTRACT FROM AUTHOR]

In SIV/HIV infection, the gastrointestinal tissue dominates as an important site because of the impact of massive mucosal CD4 depletion and immune activation-induced tissue pathology. Unlike AIDS-susceptible rhesus macaques, natural hosts do not progress to AIDS and resolve immune activation earlier. Here, we examine the role of dendritic cells (DCs) in mediating immune activation and disease progression. We demonstrate that plasmacytoid DCs (pDCs) in the blood up-regulate ß7-integrin and are rapidly recruited to the colorectum after a pathogenic SIV infection in rhesus macaques. These pDCs were capable of producing proinflammatory cytokines and primed a T cytotoxic 1 response in vitro. Consistent with the up-regulation of ß7-integrin on pDCs, in vivo blockade of α4ß7-integrin dampened pDC recruitment to the colorectum and resulted in reduced immune activation. The up-regulation of ß7-integrin expression on pDCs in the blood also was observed in HIV-infected humans but not in chronically SIV-infected sooty mangabeys that show low levels of immune activation. Our results uncover a new mechanism by which pDCs influence immune activation in colorectal tissue after pathogenic immunodeficiency virus infections. [ABSTRACT FROM AUTHOR]

Abstract: The emergence of new regulatory and pro-inflammatory immune cell subsets and cytokines dictates the need to re-examine the role of these subsets in various diseases involving the immune system. IL-17 has been recently identified as a key cytokine involved in numerous autoimmune processes. However, its role in liver autoimmune diseases remains unclear. Primary biliary cirrhosis (PBC) is characterized histologically by autoreactive CD4 and CD8 T cells surrounding damaged bile ducts. CD4+ T cells are a major source of IL-17, which compose a distinct T helper subset (Th17). Thus we set out to determine the role of IL-17 in both human and a murine model of PBC in a liver-targeted manner. Our data demonstrate an increase in the frequency of IL-17+ lymphocytic infiltration in liver tissues from PBC patients and those with other liver dysfunctions as compared to healthy livers. IL-2 receptor α knockout mice, a recently identified murine model of human PBC, also demonstrate marked aggregations of IL-17-positive cells within portal tracts and increased frequencies of Th17 cells in the liver compared to the periphery. Interestingly, CD4+ T cells from livers of normal C57BL/6J mice also secreted higher levels of IL-17 relative to those from spleens, indicating a preferential induction of Th17 cells in liver tissues. Importantly, C57BL/6J cocultures of splenic CD4+ T cells and liver non-parenchymal cells increased IL-17 production approximately 10-fold compared to T cells alone, suggesting a role of the liver microenvironment in Th17 induction in cases of liver autoimmunity and other liver inflammatory diseases. [Copyright &y& Elsevier]

Evaluation of cytokine production in macaques has been hampered by a lack of availability of optimized and standardized immunoassays such as ELISA and enzyme-linked immune spot assay (ELISpot); only a limited number of macaque cytokines have been assessed by ELISpot. Using monoclonal antibodies (mAb) to human cytokines that cross-react with cynomolgus and rhesus macaque interferon-γ (IFN-γ), interleukin (IL)-2, IL-4, IL-5, IL-6, IL-12, IL-13 and granulocyte monocyte colony-stimulating factor, we measured macaque cytokine production by ELISA and ELISpot. Quantitation of spontaneous as well as phytohemagglutinin (PHA)-induced cytokine production in peripheral blood mononuclear cells (PBMC) from rhesus and cynomolgus macaques and humans were compared. The proportional distribution of the different cytokines, in terms of PBMC synthesizing different cytokines as well as the levels of the different cytokines produced, were similar in all species. Spontaneous- and PHA-induced cytokine productions thus appear to be similarly regulated in macaques and man. ELISpot and ELISA assays for macaque IFN-γ were further used to measure antigen-specific immune responses of PBMC from cynomolgus macaques exposed to, or vaccinated against, simian immunodeficiency virus (SIV). The establishment of reliable immunoassays for detection of macaque cytokines is of importance for future progress of research utilizing macaques as experimental animals. [Copyright &y& Elsevier]