Background/Objectives: Acne vulgaris is a skin disorder that affects millions worldwide, with Cutibacterium acnes playing a key role in its inflammation. Antibiotics reduce C. acnes and inflammation, but growing antibiotic resistance has limited their efficacy. Additionally, other common acne treatments with bactericidal activity, like benzoyl peroxide, cause irritation, dryness, and peeling. To fulfill the unmet need for alternative therapies, our strategy focused on identifying potent phage lysins and/or their derived cationic peptides. Methods: The C-terminal cationic antimicrobial peptide of the Prevotella intermedia phage lysin PlyPi01 was synthesized along with several sequence-engineered variants in an attempt to enhance their bactericidal efficacy. In vitro bacterial killing assays evaluated the potency of the lysin-derived peptide derivatives against C. acnes and Staphylococcus aureus, another skin bacterium associated with acne. Antibacterial activity was assessed both in conditions simulating the human skin and in combination with retinoids. Results: The variant peptide P156 was engineered by adding arginine residues at both the N- and C-terminal ends of the parental peptide PiP01. P156 was highly potent and eradicated all tested strains of C. acnes and S. aureus. P156 acted rapidly (>5-log kill in 10 min), further reducing the potential of resistance development. Additionally, P156 maintained its potency under conditions (e.g., temperature, pH, and salt concentration) observed on the skin surface and in hair follicles, as well as in combination with retinoid—all without being toxic to human cells. Conclusions: These collective findings position P156 as a promising topical drug for clinical applications to control acne vulgaris.

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.

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 IL36 gamma 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.

Keratinocyte (KC) response to bacteria contributes to inflammatory skin diseases by induction of proinflammatory and antimicrobial responses. Recent studies show that atopic dermatitis lesions are dominated by Staphylococcus aureus (SA), whereas bacterial communities in psoriasis are diverse (Alekseyenko et al., 2013; Fyhrquist et al., 2019). Indeed, psoriasis is associated with multiple microbial species: one study reported that the combined relative abundance of Corynebacterium, Propionibacterium, Staphylococcus, and Streptococcus are increased in psoriatic epidermis (Alekseyenko et al., 2013), whereas another reported increased abundance of Corynebacterium simulans and Corynebacterium kroppenstedtii as well as Finegoldia and Neisseriaceae species (Fyhrquist et al., 2019).

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.

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-12Rβ2-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-12Rβ2-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-12Rβ2-expressing Treg subpopulation may have a therapeutic advantage in suppressing Th1 mediated autoimmunity.

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 β (TGF-β) 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.

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.

SummarySystemic 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‐γ (IFN‐γ) secretion. Moreover, DCs derived from hCDR1‐treated mice downregulated proliferation and IFN‐γ secretion by T cells from untreated mice. Upregulation of transforming growth factor‐β (TGF‐β) secretion by T cells, following treatment with hCDR1, resulted in downregulation of IFN‐γ 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‐β 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.

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 TGF beta 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.

Click to increase image sizeClick to decrease image size Acknowledgments We are grateful to Dr T. Miron for providing us with allicin.

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‐γ secretion by T cells in association with down‐regulated T‐bet expression and NF‐κB 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 (NZB×NZW)F1 mice that were treated with hCDR1. Addition of TGF‐β, 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‐β antibodies added to hCDR1‐treated T cells abrogated its effect. Thus, hCDR1 elevates TGF‐β, which contributes to the up‐regulation of T cell Foxj1 and Foxo3a expression, leading to inhibition of NF‐κB activation and IFN‐γ secretion, which is required for the maintenance of SLE.