Gram-negative anaerobes elicit a robust keratinocytes immune response with potential insights into HS pathogenesis.

Hidradenitis Suppurativa (HS) is a chronic autoinflammatory skin disease with activated keratinocytes, tunnel formation and a complex immune infiltrate in tissue. The HS microbiome is polymicrobial with an abundance of commensal gram-positive facultative (GPs) Staphylococcus species and gram-negative anaerobic (GNA) bacteria like Prevotella, Fusobacterium and Porphyromonas with increasing predominance of GNAs with disease severity. We sought to define the keratinocyte response to bacteria commonly isolated from HS lesions to probe pathogenic relationships between HS and the microbiome. Type strains of Prevotella nigrescens, Prevotella melaninogenica, Prevotella intermedia, Prevotella asaccharolytica, Fusobacterium nucleatum, as well as Staphylococcus aureus and the normal skin commensal Staphylococcus epidermidis were heat-killed and co-incubated with normal human keratinocytes. RNA was collected and analysed using RNAseq and RT-qPCR. The supernatant was collected from cell culture for protein quantification. Transcriptomic profiles between HS clinical samples and stimulated keratinocytes were compared. Co-staining of patient HS frozen sections was used to localize bacteria in lesions. A mouse intradermal injection model was used to investigate early immune recruitment. TLR4 and JAK inhibitors were used to investigate mechanistic avenues of bacterial response inhibition. GNAs, especially F. nucleatum, stimulated vastly higher CXCL8, IL17C, CCL20, IL6, TNF and IL36γ transcription in normal skin keratinocytes than the GPs S. epidermidis and S. aureus. Using RNAseq, we found that F. nucleatum (and Prevotella) strongly induced the IL-17 pathway in keratinocytes and overlapped with transcriptome profiles of HS patient clinical samples. Bacteria were juxtaposed to activated keratinocytes in vivo, and F. nucleatum strongly recruited murine neutrophil and macrophage migration. Both the TLR4 and pan-JAK inhibitors reduced cytokine production. Detailed transcriptomic profiling of healthy skin keratinocytes exposed to GNAs prevalent in HS revealed a potent, extensive inflammatory response vastly stronger than GPs. GNAs stimulated HS-relevant genes, including many genes in the IL-17 response pathway, and were significantly associated with HS tissue transcriptomes. The close association of activated keratinocytes with bacteria in HS lesions and innate infiltration in murine skin cemented GNA pathogenic potential. These novel mechanistic insights could drive future targeted therapies.

Strains of bacterial species induce a greatly varied acute adaptive immune response: The contribution of the accessory genome.

A fundamental question in human susceptibility to bacterial infections is to what extent variability is a function of differences in the pathogen species or in individual humans. To focus on the pathogen species, we compared in the same individual the human adaptive T and B cell immune response to multiple strains of two major human pathogens, Staphylococcus aureus and Streptococcus pyogenes. We found wide variability in the acute adaptive immune response induced by various strains of a species, with a unique combination of activation within the two arms of the adaptive response. Further, this was also accompanied by a dramatic difference in the intensity of the specific protective T helper (Th) response. Importantly, the same immune response differences induced by the individual strains were maintained across multiple healthy human donors. A comparison of isogenic phage KO strains, demonstrated that of the pangenome, prophages were the major contributor to inter-strain immune heterogeneity, as the T cell response to the remaining "core genome" was noticeably blunted. Therefore, these findings extend and modify the notion of an adaptive response to a pathogenic bacterium, by implying that the adaptive immune response signature of a bacterial species should be defined either per strain or alternatively to the species' 'core genome', common to all of its strains. Further, our results demonstrate that the acquired immune response variation is as wide among different strains within a single pathogenic species as it is among different humans, and therefore may explain in part the clinical heterogeneity observed in patients infected with the same species.

Quantifying how much host, pathogen, and other factors affect human protective adaptive immune responses.

Recognizing the "essential" factors that contribute to a clinical outcome is critical for designing appropriate therapies and prioritizing limited medical resources. Demonstrating a high correlation between a factor and an outcome does not necessarily imply an essential role of the factor to the outcome. Human protective adaptive immune responses to pathogens vary among (and perhaps within) pathogenic strains, human individual hosts, and in response to other factors. Which of these has an "essential" role? We offer three statistical approaches that predict the presence of newly contributing factor(s) and then quantify the influence of host, pathogen, and the new factors on immune responses. We illustrate these approaches using previous data from the protective adaptive immune response (cellular and humoral) by human hosts to various strains of the same pathogenic bacterial species. Taylor's law predicts the existence of other factors potentially contributing to the human protective adaptive immune response in addition to inter-individual host and intra-bacterial species inter-strain variability. A mixed linear model measures the relative contribution of the known variables, individual human hosts and bacterial strains, and estimates the summed contributions of the newly predicted but unknown factors to the combined adaptive immune response. A principal component analysis predicts the presence of sub-variables (currently not defined) within bacterial strains and individuals that may contribute to the combined immune response. These observations have statistical, biological, clinical, and therapeutic implications.

The role of dendritic cells in the mechanism of action of a peptide that ameliorates lupus in murine models.

Systemic lupus erythematosus (SLE) is characterized in its early stages by the expansion of autoreactive T cells that trigger B-cell activation with subsequent multi-organ injury. Dendritic cells (DCs) in lupus were found to display an aberrant phenotype with higher expression of the maturation markers major histocompatibility complex (MHC) class II, CD80 and CD86, as well as higher production of proinflammatory cytokines including interleukin-12 (IL-12), resulting in an increased ability to activate T cells. A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorated SLE in both induced and spontaneous lupus models by downregulating T-cell functions. Our objectives were to determine whether DCs play a role in promoting the beneficial effects of hCDR1. We showed here that treatment with hCDR1 lowered the expression levels of MHC class II, CD80 and CD86 on DCs. The latter effect was associated with downregulation of messenger RNA expression and secretion of IL-12, a cytokine that upregulated T-cell proliferation and interferon-gamma (IFN-gamma) secretion. Moreover, DCs derived from hCDR1-treated mice downregulated proliferation and IFN-gamma secretion by T cells from untreated mice. Upregulation of transforming growth factor-beta (TGF-beta) secretion by T cells, following treatment with hCDR1, resulted in downregulation of IFN-gamma production and contributed to the phenotypic changes and magnitude of IL-12 secretion by DCs. The ameliorating effects of hCDR1 are therefore mediated at least partially by the upregulated secretion of TGF-beta by T cells that contribute to the induction of DCs with immature phenotype and suppressed functions. The resulting DCs further downregulate autoreactive T-cell functions.

Dendritic Cells Induce a Subpopulation of IL-12Rß2-Expressing Treg that Specifically Consumes IL-12 to Control Th1 Responses.

Cytokines secreted from dendritic cells (DCs) play an important role in the regulation of T helper (Th) cell differentiation and activation into effector cells. Therefore, controlling cytokine secretion from DCs may potentially regulate Th differentiation/activation. DCs also induce de-novo generation of regulatory T cells (Treg) that modulate the immune response. In the current study we used the mixed leukocyte reaction (MLR) to investigate the effect of allospecific Treg on IL-12, TNFα and IL-6 secretion by DCs. Treg cells were found to markedly down-regulate IL-12 secretion from DCs following stimulation with TLR7/8 agonist. This down-regulation of IL-12 was neither due to a direct suppression of its production by the DCs nor a result of marked DC death. We found that IL-12 was rather actively consumed by Treg cells. IL-12 consumption was mediated by a subpopulation of IL-12Rß2-expressing Treg cells and was dependent on MHC class-II expressed on dendritic cells. Furthermore, IL-12 consumption by Tregs increased their suppressive effect on T cell proliferation and Th1 activation. These results provide a new pathway of Th1 response regulation where IL-12 secreted by DCs is consumed by a sub-population of IL-12Rß2-expressing Treg cells. Consumption of IL-12 by Tregs not only reduces the availability of IL-12 to Th effector cells but also enhances the Treg immunosuppressive effect. This DC-induced IL-12Rß2-expressing Treg subpopulation may have a therapeutic advantage in suppressing Th1 mediated autoimmunity.

Dendritic cells induce antigen-specific regulatory T cells that prevent graft versus host disease and persist in mice.

Foxp3(+) regulatory T cells (T reg cells) effectively suppress immunity, but it is not determined if antigen-induced T reg cells (iT reg cells) are able to persist under conditions of inflammation and to stably express the transcription factor Foxp3. We used spleen cells to stimulate the mixed leukocyte reaction (MLR) in the presence of transforming growth factor ß (TGF-ß) and retinoic acid. We found that the CD11c(high) dendritic cell fraction was the most potent at inducing high numbers of alloreactive Foxp3(+) cells. The induced CD4(+)CD25(+)Foxp3(+) cells appeared after extensive proliferation. When purified from the MLR, iT reg cells suppressed both primary and secondary MLR in vitro in an antigen-specific manner. After transfer into allogeneic mice, iT reg cells persisted for 6 mo and prevented graft versus host disease (GVHD) caused by co-transferred CD45RB(hi) T cells. Similar findings were made when iT reg cells were transferred after onset of GVHD. The CNS2 intronic sequence of the Foxp3 gene in the persisting iT reg cells was as demethylated as the corresponding sequence of naturally occurring T reg cells. These results indicate that induced Foxp3(+) T reg cells, after proliferating and differentiating into antigen-specific suppressive T cells, can persist for long periods while suppressing a powerful inflammatory disease.

The tolerogenic peptide hCDR1 downregulates pathogenic cytokines and apoptosis and upregulates immunosuppressive molecules and regulatory T cells in peripheral blood mononuclear cells of lupus patients.

A tolerogenic peptide, hCDR1, ameliorated murine lupus via the upregulation of functional regulatory cells and by immunomodulating cytokine production. In the present study we analyzed the ability of hCDR1 to similarly affect gene expression and regulatory T cells when incubated with peripheral blood mononuclear cells (PBMC) of lupus patients. To this end, peripheral blood mononuclear cells (PBMC) of 11 lupus patients and five gender- and age-matched healthy controls were cultured with hCDR1 or a control peptide. Gene expression and regulatory T-cells were assessed. hCDR1 significantly downregulated interleukin (IL)-1beta, interferon (IFN)-gamma, and IL-10 gene expression. Furthermore, hCDR1 upregulated the expression of the anti-apoptotic Bcl-xL molecule and downregulated the pro-apoptotic caspase-3, resulting in reduced rates of apoptosis. hCDR1 increased the expression of transforming growth factor (TGF)-beta, FoxP3 and the negative regulators Foxj1 and Foxo3a. No significant effects were observed using a control peptide or when PBMC of healthy donors were incubated with hCDR1. The elevated gene expression of FoxP3 was due to hCDR1-induced upregulation of TGF-beta, resulting in an increase of CD4+CD25+FoxP3+ functional, regulatory cells. The ability of the regulatory cells to diminish IFN-gamma expression and to upregulate TGF-beta was abrogated after the addition of a neutralizing anti-CD25 antibody, confirming their role in the beneficial effects of hCDR1.

A peptide that ameliorates lupus up-regulates the diminished expression of early growth response factors 2 and 3.

Expansion of autoreactive T cells and their resistance to anergy was demonstrated in systemic lupus erythematosus (SLE). A pair of transcription factors, early growth response 2 (Egr-2) and 3 (Egr-3), are negative regulators of T cell activation that were shown to be important in anergy. A peptide (designated hCDR1 for human CDR1) based on the CDR-1 of an anti-DNA Ab ameliorated SLE in both induced and spontaneous lupus models. Our objectives were to determine the expression levels of Egr-2 and Egr-3 in autoreactive T cells following immunization with the lupus-inducing anti-DNA Ab that bears a common Id designated 16/6Id and also in a full-blown SLE and to determine the effect of hCDR1 on these transcription factors. We demonstrated diminished expression levels of Egr-2 and Egr-3 mRNA both early after immunization with the 16/6Id and in SLE-afflicted (NZB x NZW)F1 (New Zealand Black and New Zealand White) mice. Furthermore, by down-regulating Akt phosphorylation and up-regulating TGFbeta secretion, treatment with hCDR1 significantly up-regulated Egr-2 and Egr-3 expression. This was associated with an increased expression of the E3 ligase Cbl-b. Inhibition of Akt in T cells of immunized mice decreased, whereas silencing of the Egr-2 and Egr-3 in T cells of hCDR1-treated mice increased IFN-gamma secretion. Thus, hCDR1 down-regulates Akt phosphorylation, which leads to up-regulated expression of T cell Egr-2 and Egr-3, resulting in the inhibition of IFN-gamma secretion that is required for the maintenance of SLE.

The negative regulators Foxj1 and Foxo3a are up-regulated by a peptide that inhibits systemic lupus erythematosus-associated T cell responses.

A peptide (hCDR1) based on the complementarity determining region-1 of an anti-DNA antibody ameliorates systemic lupus erythematosus (SLE) in induced and spontaneous lupus models. Our objectives were to determine the effects of hCDR1 on TCR signaling and on its negative regulators, Foxj1 and Foxo3a. BALB/c mice were immunized with the SLE-inducing anti-DNA antibody, designated 16/6Id, and treated with hCDR1. hCDR1 treatment specifically inhibited IFN-gamma secretion by T cells in association with down-regulated T-bet expression and NF-kappaB activation; however, GATA-3 expression was not affected. Furthermore, TCR signaling (ZAP-70 phosphorylation) was inhibited, and the mRNA expression of the two modulators of Th1 activation, Foxj1 and Foxo3a, was significantly up-regulated. The latter were also elevated in SLE-afflicted (NZBxNZW)F1 mice that were treated with hCDR1. Addition of TGF-beta, which was elevated following treatment with hCDR1, to T cells from 16/6Id immunized mice, up-regulated Foxj1 and Foxo3a mRNA expression, similarly to hCDR1. In contrast, anti-TGF-beta antibodies added to hCDR1-treated T cells abrogated its effect. Thus, hCDR1 elevates TGF-beta, which contributes to the up-regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF-kappaB activation and IFN-gamma secretion, which is required for the maintenance of SLE.

Down-regulation of stromal cell-derived factor-1alpha-induced T cell chemotaxis by a peptide based on the complementarity-determining region 1 of an anti-DNA autoantibody via up-regulation of TGF-beta secretion.

Systemic lupus erythematosus (SLE) can be induced in mice by immunizing them with a monoclonal human anti-DNA Ab that expresses a major Id, designated 16/6Id. In addition, a peptide based on the sequence of the CDR 1 (hCDR1) of the 16/6Id ameliorated the clinical manifestations of SLE in experimental models. In this study we examined the effects of treating mice with human complementary-determining region 1 (hCDR1) on the subsequent chemotaxis of T cells derived from 16/6Id-primed mice. First we demonstrated elevated levels of stromal cell-derived factor-1alpha (SDF-1alpha) in the sera of SLE-afflicted mice and in the sera and lymphoid tissues of 16/6Id-immunized BALB/c mice shortly after the immunization. We then found that administration of hCDR1 to 16/6Id-immunized mice specifically down-regulated SDF1alpha-induced T cell chemotaxis through fibronectin and collagen type I. This was accompanied by diminished SDF1-alpha-induced T cell adhesion and ERK phosphorylation. Treatment with hCDR1 up-regulated TGF-beta secretion, which, in turn, inhibited the murine T cell adhesion to and chemotaxis through fibronectin as well as their ERK phosphorylation. Thus, the secretion of TGF-beta after treatment of 16/6Id-immunized mice with hCDR1 plays an important role in the down-regulation of SDF-1alpha-mediated T cell activation and the interactions with extracellular matrix moieties observed in the present study.

The inhibition of autoreactive T cell functions by a peptide based on the CDR1 of an anti-DNA autoantibody is via TGF-beta-mediated suppression of LFA-1 and CD44 expression and function.

Systemic lupus erythematosus (SLE), which is characterized by the increased production of autoantibodies and defective T cell responses, can be induced in mice by immunization with a human anti-DNA mAb that expresses a major Id, designated 16/6Id. A peptide based on the sequence of the CDR1 of the 16/6Id (human CDR1 (hCDR1)) ameliorated the clinical manifestations of SLE and down-regulated, ex vivo, the 16/6Id-induced T cell proliferation. In this study, we examined the mechanism responsible for the hCDR1-induced modulation of T cell functions related to the pathogenesis of SLE. We found that injection of hCDR1 into BALB/c mice concomitant with their immunization with 16/6Id resulted in a marked elevation of TGF-beta secretion 10 days later. Addition of TGF-beta suppressed the 16/6Id-stimulated T cell proliferation similarly to hCDR1. In addition, we provide evidence that one possible mechanism underlying the hCDR1- and TGFbeta-induced inhibition of T cell proliferation is by down-regulating the expression, and therefore the functions, of a pair of key cell adhesion receptors, LFA-1 (alphaLbeta2) and CD44, which operate as accessory molecules in mediating APC-T cell interactions. Indeed, T cells of mice treated with hCDR1 showed a TGF-beta-induced suppression of adhesion to the LFA-1 and CD44 ligands, hyaluronic acid and ICAM-1, respectively, induced by stromal cell-derived factor-1alpha and PMA. The latter suppression is through the inhibition of ERK phosphorylation. Thus, the down-regulation of SLE-associated responses by hCDR1 treatment may be due to the effect of the up-regulated TGF-beta on the expression and function of T cell adhesion receptors and, consequently, on T cell stimulation, adhesion, and proliferation.

Allicin inhibits SDF-1alpha-induced T cell interactions with fibronectin and endothelial cells by down-regulating cytoskeleton rearrangement, Pyk-2 phosphorylation and VLA-4 expression.

Allicin, a major ingredient of fresh garlic extract that is produced during the crushing of garlic cloves, exerts various beneficial biological effects, including a broad spectrum of antimicrobial activity, antihyperlipidaemic and antihypertensive effects. However, how allicin affects the immune system is less well known, and its effect on human T cells has never been studied. Here, we examined the in-vitro effects of allicin on the functioning of T cells related to their entry to inflamed extravascular sites. We found that allicin (20-100 microm) inhibits the SDF-1alpha (CXCL12)-induced T cell migration through fibronectin (FN), and that this inhibition is mediated by the down-regulation of (i) the reorganization of cortical actin and the subsequent T cell polarization, and (ii) T cell adhesion to FN. Moreover, allicin also inhibited T cell adhesion to endothelial cells and transendothelial migration. The mechanisms underlying these inhibitory effects of allicin are associated with its ability to down-regulate the phosphorylation of Pyk2, an intracellular member of the focal adhesion kinases, and to reduce the expression of the VCAM-1- and FN-specific alpha4beta1-integrin (VLA-4). The ability of allicin to down-regulate these chemokine-induced and VLA-4-mediated T cell functions explains its beneficial biological effects in processes where T cells play an important role and suggests that allicin may be used therapeutically with chronic inflammatory diseases.