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635 NF-κB activation and psoriasiform inflammation by Piasy deficiency
Innate Immunity, Microbiology, Inflammation | ABSTRACTS 630
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Neutrophils are a major source of IL-17A in psoriatic skin and can produce IL-17F and IL-22 B Dyring-Andersen1, T Velte Honore´2, A Madelung3, M Bzorek4, S Simonsen2, S Novrup Clemmensen5, RA Clark6, N Borregaard5 and L Skov2 1 Brigham and Women’s Hospital and HMS, Boston, MA, 2 Gentofte Hospital, Copenhagen, Hovedstaden, Denmark, 3 Department of Pathology, Rigshospitalet, Copenhagen, Hovedstaden, Denmark, 4 Zealand University Hospital, Naestved, Sjelland, Denmark, 5 Rigshospitalet, Copenhagen, Hovedstaden, Denmark and 6 Brigham and Women’s Hospital and HMS, Boston, MA Psoriasis is a T cell and IL-17 dependent inflammatory skin disease. Helper T cells have been assumed to be the major source of IL-17 in psoriasis but other cell types can also produce this cytokine. We immunostained human psoriatic lesions (n¼15) and healthy skin (n¼10) for IL17A, T cells (CD4, CD8) and neutrophils (myeloperoxidase, MPO). We found that 32% of MPO+ neutrophils in psoriasis samples produced IL-17A, compared with less than 1% of total CD4+ and CD8+ cells. There were on average 10.6 IL-17A producing neutrophils per 10 high power fields in psoriasis, compared to 2.7 IL-17A producing T cells (p¼0.008). IL-17A producing neutrophils outnumbered IL-17A producing T cells four-fold demonstrating that neutrophils are an important source of IL-17A in psoriasis. To explore potential interactions between keratinocytes and neutrophils in psoriasis, we co-cultured these cells in vitro and studied neutrophil cytokine production by quantitative RT-PCR and intracellular immunostaining and flow cytometry analysis. Neutrophils co-cultured with keratinocytes upregulated production of IL-17A, IL-17F and IL-22 at both the protein and RNA levels. Neutrophils cultured with keratinocytes lost CD62L and upregulated CD11b, consistent with activation. In summary, this study suggests that neutrophils, known for long to be a part of the histologic landscape of psoriasis, are a major source of IL-17A production and have the potential to contribute to inflammation by producing IL-17F and IL-22. This is, to our knowledge, the first report that human neutrophils can produce IL-17F and IL-22.
TLR2/MyD88 signaling on T cells mediates a compensatory protective immune response to IL-1b/MyD88 signaling against secondary S. aureus skin challenge C Dillen1, B Pinsker2, H Liu3, Y Wang1, R Ortines1, N Archer1 and L Miller4 1 Johns Hopkins Medicine, Baltimore, MD, 2 Johns Hopkins University, Baltimore, MD, 3 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD and 4 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD The immune responses that mediate long-lasting protection against community-acquired methicillin-resistant S. aureus (CA-MRSA) skin reinfections are unclear. Using a CA-MRSA skin reinfection model (USA300 LAC::lux; 3x107 CFU, intradermally) involving a primary infection (1 ) in the lower back (which cleared by 14 days) followed by a secondary infection (2 ) in the upper back on day 28, we previously reported that 1 IL-1b-/- mice developed larger lesions, increased bacterial burden and impaired neutrophil recruitment than wt mice whereas 2 IL-1b-/- mice were completely protected. To evaluate which cell types mediated the protection, cre/lox mice were generated in which MyD88 was specifically deleted in keratinocytes (K14-creERT2), myeloid cells (LysM-cre) or T cells (Lck-cre) and only 1 and 2 Lck-CreMyD88fl/fl mice developed larger lesions and increased bacterial burden compared with wt mice. Since 2 Lck-CreMyD88fl/fl mice but not 2 IL-1b-/- mice developed protection, this suggests that an alternative MyD88 signal on T cells provided a compensatory protective immune response. We hypothesized either IL-1a, which like IL-1b also signals via IL-1R/MyD88, or TLR2, which recognizes S. aureus components, could represent this alternative MyD88 signal. IL-1b-/- mice treated with an IL-1R blocking antibody (to additionally block IL-1a) were similarly protected during the 2 infection, indicating that IL-1a was not involved. In contrast, TLR2-/- mice treated with an IL-1R blocking antibody resulted in complete loss of the protection during the 2 infection. Taken together, IL-1b is important for immunity during a 1 S. aureus skin infection whereas TLR2 signaling on T cells provided a MyD88-dependent compensatory response for protection against a secondary S. aureus skin challenge.
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IL-36b provides protection against herpes simplex virus-1 K Milora, S Uppalapati and LE Jensen Temple University School of Medicine, Philadelphia, PA Interleukin-36 (IL-36) cytokines have been associated with plaque psoriasis and generalized pustular psoriasis. There are three genes encoding distinct IL-36 cytokines, IL-36a, IL-36b and IL-36g. Using knockout (KO) mice for each of the individual IL-36 genes, we previously showed that psoriasis-like skin inflammation induced by imiquimod is dependent upon IL36a, but not IL-36b and IL-36g. Imiquimod is an antiviral drug that sometimes is used to treat skin disease caused by acyclovir resistant herpes simplex virus-1 (HSV-1). Here we employed the flank skin HSV-1 infection mouse model to examine the role of the IL-36 cytokines in restricting HSV-1 progression. Surprisingly, despite the involvement of IL-36a in imiquimod induced skin inflammation, the absence of IL-36a did not affect the outcome of HSV-1 skin infection. In contrast, mice deficient of IL-36b developed up to 3-times larger (p < 0.001) skin lesions than wildtype mice. In addition, while IL-36a and IL-36g KO mice exhibited the same survival rate (approx. 75%) as wildtype mice, only approx. 25% of IL-36b deficient mice survived (p < 0.001). Cell culture studies have implicated IL-36 in initiation of adaptive immune responses and production of the antiviral IFN-g by CD4+ cells. Such responses could potentially explain the outcome of our in vivo results. Against expectations, in vivo development of HSV-1 specific antibodies and CD8+cells did not require IL-36b. Using ELISpot assays, we also found numbers of IFN-g producing CD4 cells in HSV-1 infected IL-36b KO skin to be the same as in wildtype mice at day 6 post-infection. Viral titers in the primary lesions were the same at days 3 and 5 in both wildtype and IL-36b KO mice. At day 7 viral titers started to decreased (approx. 200-fold, p < 0.01); however, no difference between wildtype and IL-36b KO mice were detected. Our data demonstrate equal adaptive immune responses in wildtype and IL-36b KO mice during HSV-1 infection, and suggest a novel role for IL-36b in protecting the skin from HSV-1. This may have implications upon the development of new therapies for psoriasis, e.g., strategies targeting IL-36 signaling.
Aging and diet-induced obesity impair activation of adipocytes that protect against invasive Staphylococcus aureus skin infection L Zhang1, F Li2 and RL Gallo2 1 University of California, San Diego, San Diego, CA and 2 UCSD, San Diego, CA Staphylococcus aureus is responsible for the majority of skin and soft tissue infections, and aging and the growing obesity epidemic have further increased this risk. We recently discovered that dermal white adipose tissue (DWAT) protects against invasive S. aureus infection by producing the antimicrobial peptide cathelicidin (CAMP). Here we investigated if aging and obesity may disturb this beneficial immune response from DWAT. Using mouse models of aging and high fat diet-induced obesity (DIO), we found that aging and DIO led to thinning of the dermis and terminal differentiation of adipogenic fibroblasts to mature adipocytes. FACS analyses and primary culture of dermal fibroblasts confirmed that aging was associated with loss of adipogenic fibroblasts in the dermis, and DIO further shifted these dermal fibroblasts to a phenotype with greatly reduced capacity to produce CAMP during adipogenesis. As a result, pathogen-triggered adipogenesis responses, including preadipocyte activation and CAMP production, were greatly impaired in aged mice compared to young controls, and in obese compared to normal diet mice. These diminished DWAT functions seen in aged and/or DIO mice correlated with increased susceptibility to S. aureus infection. Furthermore, we found stimulation of adipogenesis by a PPARg agonist Rosiglitazone restored the host defense function of DIO mice, and decreased their susceptibility to S. aureus. The beneficial effect of Rosiglitazone was partially dependent on Camp expression in adipocytes as Rosiglitazone was not as effective in adipocyte-specific Camp knockout mice. Together, our results suggest that DWAT dysfunction may be responsible for the loss of skin defense in aging and/or obesity. These observations further emphasize the clinical significance of cathelicidin expression in DWAT, and suggest a potential novel therapeutic approach for treatment of S. aureus skin infection with Rosiglitazone.
Role of the cutaneous microbiome in the pathogenesis of psoriasis D Yan, H Chang, R Singh, K Lai, L Afifi, X Lu and W Liao University of California, San Francisco, Department of Dermatology, San Francisco, CA Psoriasis is a chronic, inflammatory skin disease that affects 2-3 percent of the US population, yet its pathogenesis is poorly understood. The cutaneous microbiome’s interplay with the host immune system suggests that it may contribute to the development of psoriasis in genetically predisposed individuals. This study aims to characterize the psoriatic skin microbiome and understand its role in psoriasis pathogenesis. 16s rRNA sequencing of site-matched skin swabs from 8 psoriasis patients and 8 healthy controls revealed reduced alpha diversity in lesional psoriasis skin compared to controls (402 OTU’s vs. 578 OTU’s, p¼0.04). At the phylum level, lesional skin had higher Firmicutes (p¼0.009) and less Actinobacteria (p¼0.0001) compared to controls. At the genus level, lesional skin had more Alloiococcus (p¼ 0.01) and Aerococcus (p¼ 0.01) and demonstrated a trend towards lower Propionibacterium (p¼0.08) and higher Gallicola (p¼0.09) compared to controls. Interestingly, Alloiococcus (p¼0.003) and Gallicola (p¼0.04) were also higher in non-lesional skin compared to controls. Furthermore, lesional and non-lesional skin shared an increased abundance of Acinetobacter sp., Staphylococcus pettenkoferi, and Streptococcus sp., relative to controls. Lesional and non-lesional psoriasis skin did not differ significantly in microbiome composition. These data suggest intriguing differences in the cutaneous microbiome of psoriatic individuals and healthy controls that may advance our understanding of psoriasis pathogenesis.
NF-kB activation and psoriasiform inflammation by Piasy deficiency Y Liu1, R De La Torre1, R Bridges1, E Swanzey1, K Olsen1, Z Wang1 and M Kulesz-Martin2 1 Oregon Health and Science University, Portland, OR and 2 OHSU, Portland, OR NF-kB is one of the major contributors in the inflammation circuitry of psoriasis. It mediates the signal transduction of many psoriatic cytokines including TNFa and IL-17. Despite the identification of psoriasis associated SNPs in genes involved in NF-kB signaling pathways, the molecular mechanism of aberrant NF-kB activation in psoriasis remains largely unknown. In this study, we have provided experimental evidence to suggest that the inactivation of PIASy contributes to constitutive NF-kB activation and psoriasis pathogenesis. PIASy-deficient mice developed exacerbated psoriasis-like phenotypes with sustained NF-kB activation in response to topical imiquimod treatment. PIASy deficiency resulted in robust expression of psoriatic cytokines and chemokines from keratinocytes in response to TNFa and IL-17A. Biochemically, we have identified Piasy as a NF-kB associated protein. Piasy protein bound to the NFkB RelA subunit through its C-terminus. Piasy negatively regulated NF-kB activity. Negative regulation of NF-kB was not mediated through its E3 SUMO ligase activity. Piasy inhibited RelA DNA binding in vitro measured by DNA affinity immunoblotting. Piasy protein also bound to the NF-kB binding sites in the CCL20 promoter in vivo as detected by ChIP assay. These lines of evidence suggest that Piasy represses NF-kB activity through inhibiting RelA DNA binding. PIASy is a transcriptional repressor that localizes in the nucleus. Immunostaining analysis of PIASy in psoriatic skin revealed that PIASy cytoplasmic localization was elevated in psoriatic compared to healthy epidermis. Taken together, it suggests that PIASy negatively regulates NF-kB and PIASy inactivation contributes to psoriasis pathogenesis.
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Neutrophils are a major source of IL-17A in psoriatic skin and can produce IL-17F and IL-22 B Dyring-Andersen1, T Velte Honore´2, A Madelung3, M Bzorek4, S Simonsen2, S Novrup Clemmensen5, RA Clark6, N Borregaard5 and L Skov2 1 Brigham and Women’s Hospital and HMS, Boston, MA, 2 Gentofte Hospital, Copenhagen, Hovedstaden, Denmark, 3 Department of Pathology, Rigshospitalet, Copenhagen, Hovedstaden, Denmark, 4 Zealand University Hospital, Naestved, Sjelland, Denmark, 5 Rigshospitalet, Copenhagen, Hovedstaden, Denmark and 6 Brigham and Women’s Hospital and HMS, Boston, MA Psoriasis is a T cell and IL-17 dependent inflammatory skin disease. Helper T cells have been assumed to be the major source of IL-17 in psoriasis but other cell types can also produce this cytokine. We immunostained human psoriatic lesions (n¼15) and healthy skin (n¼10) for IL17A, T cells (CD4, CD8) and neutrophils (myeloperoxidase, MPO). We found that 32% of MPO+ neutrophils in psoriasis samples produced IL-17A, compared with less than 1% of total CD4+ and CD8+ cells. There were on average 10.6 IL-17A producing neutrophils per 10 high power fields in psoriasis, compared to 2.7 IL-17A producing T cells (p¼0.008). IL-17A producing neutrophils outnumbered IL-17A producing T cells four-fold demonstrating that neutrophils are an important source of IL-17A in psoriasis. To explore potential interactions between keratinocytes and neutrophils in psoriasis, we co-cultured these cells in vitro and studied neutrophil cytokine production by quantitative RT-PCR and intracellular immunostaining and flow cytometry analysis. Neutrophils co-cultured with keratinocytes upregulated production of IL-17A, IL-17F and IL-22 at both the protein and RNA levels. Neutrophils cultured with keratinocytes lost CD62L and upregulated CD11b, consistent with activation. In summary, this study suggests that neutrophils, known for long to be a part of the histologic landscape of psoriasis, are a major source of IL-17A production and have the potential to contribute to inflammation by producing IL-17F and IL-22. This is, to our knowledge, the first report that human neutrophils can produce IL-17F and IL-22.
TLR2/MyD88 signaling on T cells mediates a compensatory protective immune response to IL-1b/MyD88 signaling against secondary S. aureus skin challenge C Dillen1, B Pinsker2, H Liu3, Y Wang1, R Ortines1, N Archer1 and L Miller4 1 Johns Hopkins Medicine, Baltimore, MD, 2 Johns Hopkins University, Baltimore, MD, 3 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD and 4 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD The immune responses that mediate long-lasting protection against community-acquired methicillin-resistant S. aureus (CA-MRSA) skin reinfections are unclear. Using a CA-MRSA skin reinfection model (USA300 LAC::lux; 3x107 CFU, intradermally) involving a primary infection (1 ) in the lower back (which cleared by 14 days) followed by a secondary infection (2 ) in the upper back on day 28, we previously reported that 1 IL-1b-/- mice developed larger lesions, increased bacterial burden and impaired neutrophil recruitment than wt mice whereas 2 IL-1b-/- mice were completely protected. To evaluate which cell types mediated the protection, cre/lox mice were generated in which MyD88 was specifically deleted in keratinocytes (K14-creERT2), myeloid cells (LysM-cre) or T cells (Lck-cre) and only 1 and 2 Lck-CreMyD88fl/fl mice developed larger lesions and increased bacterial burden compared with wt mice. Since 2 Lck-CreMyD88fl/fl mice but not 2 IL-1b-/- mice developed protection, this suggests that an alternative MyD88 signal on T cells provided a compensatory protective immune response. We hypothesized either IL-1a, which like IL-1b also signals via IL-1R/MyD88, or TLR2, which recognizes S. aureus components, could represent this alternative MyD88 signal. IL-1b-/- mice treated with an IL-1R blocking antibody (to additionally block IL-1a) were similarly protected during the 2 infection, indicating that IL-1a was not involved. In contrast, TLR2-/- mice treated with an IL-1R blocking antibody resulted in complete loss of the protection during the 2 infection. Taken together, IL-1b is important for immunity during a 1 S. aureus skin infection whereas TLR2 signaling on T cells provided a MyD88-dependent compensatory response for protection against a secondary S. aureus skin challenge.
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IL-36b provides protection against herpes simplex virus-1 K Milora, S Uppalapati and LE Jensen Temple University School of Medicine, Philadelphia, PA Interleukin-36 (IL-36) cytokines have been associated with plaque psoriasis and generalized pustular psoriasis. There are three genes encoding distinct IL-36 cytokines, IL-36a, IL-36b and IL-36g. Using knockout (KO) mice for each of the individual IL-36 genes, we previously showed that psoriasis-like skin inflammation induced by imiquimod is dependent upon IL36a, but not IL-36b and IL-36g. Imiquimod is an antiviral drug that sometimes is used to treat skin disease caused by acyclovir resistant herpes simplex virus-1 (HSV-1). Here we employed the flank skin HSV-1 infection mouse model to examine the role of the IL-36 cytokines in restricting HSV-1 progression. Surprisingly, despite the involvement of IL-36a in imiquimod induced skin inflammation, the absence of IL-36a did not affect the outcome of HSV-1 skin infection. In contrast, mice deficient of IL-36b developed up to 3-times larger (p < 0.001) skin lesions than wildtype mice. In addition, while IL-36a and IL-36g KO mice exhibited the same survival rate (approx. 75%) as wildtype mice, only approx. 25% of IL-36b deficient mice survived (p < 0.001). Cell culture studies have implicated IL-36 in initiation of adaptive immune responses and production of the antiviral IFN-g by CD4+ cells. Such responses could potentially explain the outcome of our in vivo results. Against expectations, in vivo development of HSV-1 specific antibodies and CD8+cells did not require IL-36b. Using ELISpot assays, we also found numbers of IFN-g producing CD4 cells in HSV-1 infected IL-36b KO skin to be the same as in wildtype mice at day 6 post-infection. Viral titers in the primary lesions were the same at days 3 and 5 in both wildtype and IL-36b KO mice. At day 7 viral titers started to decreased (approx. 200-fold, p < 0.01); however, no difference between wildtype and IL-36b KO mice were detected. Our data demonstrate equal adaptive immune responses in wildtype and IL-36b KO mice during HSV-1 infection, and suggest a novel role for IL-36b in protecting the skin from HSV-1. This may have implications upon the development of new therapies for psoriasis, e.g., strategies targeting IL-36 signaling.
Aging and diet-induced obesity impair activation of adipocytes that protect against invasive Staphylococcus aureus skin infection L Zhang1, F Li2 and RL Gallo2 1 University of California, San Diego, San Diego, CA and 2 UCSD, San Diego, CA Staphylococcus aureus is responsible for the majority of skin and soft tissue infections, and aging and the growing obesity epidemic have further increased this risk. We recently discovered that dermal white adipose tissue (DWAT) protects against invasive S. aureus infection by producing the antimicrobial peptide cathelicidin (CAMP). Here we investigated if aging and obesity may disturb this beneficial immune response from DWAT. Using mouse models of aging and high fat diet-induced obesity (DIO), we found that aging and DIO led to thinning of the dermis and terminal differentiation of adipogenic fibroblasts to mature adipocytes. FACS analyses and primary culture of dermal fibroblasts confirmed that aging was associated with loss of adipogenic fibroblasts in the dermis, and DIO further shifted these dermal fibroblasts to a phenotype with greatly reduced capacity to produce CAMP during adipogenesis. As a result, pathogen-triggered adipogenesis responses, including preadipocyte activation and CAMP production, were greatly impaired in aged mice compared to young controls, and in obese compared to normal diet mice. These diminished DWAT functions seen in aged and/or DIO mice correlated with increased susceptibility to S. aureus infection. Furthermore, we found stimulation of adipogenesis by a PPARg agonist Rosiglitazone restored the host defense function of DIO mice, and decreased their susceptibility to S. aureus. The beneficial effect of Rosiglitazone was partially dependent on Camp expression in adipocytes as Rosiglitazone was not as effective in adipocyte-specific Camp knockout mice. Together, our results suggest that DWAT dysfunction may be responsible for the loss of skin defense in aging and/or obesity. These observations further emphasize the clinical significance of cathelicidin expression in DWAT, and suggest a potential novel therapeutic approach for treatment of S. aureus skin infection with Rosiglitazone.
Role of the cutaneous microbiome in the pathogenesis of psoriasis D Yan, H Chang, R Singh, K Lai, L Afifi, X Lu and W Liao University of California, San Francisco, Department of Dermatology, San Francisco, CA Psoriasis is a chronic, inflammatory skin disease that affects 2-3 percent of the US population, yet its pathogenesis is poorly understood. The cutaneous microbiome’s interplay with the host immune system suggests that it may contribute to the development of psoriasis in genetically predisposed individuals. This study aims to characterize the psoriatic skin microbiome and understand its role in psoriasis pathogenesis. 16s rRNA sequencing of site-matched skin swabs from 8 psoriasis patients and 8 healthy controls revealed reduced alpha diversity in lesional psoriasis skin compared to controls (402 OTU’s vs. 578 OTU’s, p¼0.04). At the phylum level, lesional skin had higher Firmicutes (p¼0.009) and less Actinobacteria (p¼0.0001) compared to controls. At the genus level, lesional skin had more Alloiococcus (p¼ 0.01) and Aerococcus (p¼ 0.01) and demonstrated a trend towards lower Propionibacterium (p¼0.08) and higher Gallicola (p¼0.09) compared to controls. Interestingly, Alloiococcus (p¼0.003) and Gallicola (p¼0.04) were also higher in non-lesional skin compared to controls. Furthermore, lesional and non-lesional skin shared an increased abundance of Acinetobacter sp., Staphylococcus pettenkoferi, and Streptococcus sp., relative to controls. Lesional and non-lesional psoriasis skin did not differ significantly in microbiome composition. These data suggest intriguing differences in the cutaneous microbiome of psoriatic individuals and healthy controls that may advance our understanding of psoriasis pathogenesis.
NF-kB activation and psoriasiform inflammation by Piasy deficiency Y Liu1, R De La Torre1, R Bridges1, E Swanzey1, K Olsen1, Z Wang1 and M Kulesz-Martin2 1 Oregon Health and Science University, Portland, OR and 2 OHSU, Portland, OR NF-kB is one of the major contributors in the inflammation circuitry of psoriasis. It mediates the signal transduction of many psoriatic cytokines including TNFa and IL-17. Despite the identification of psoriasis associated SNPs in genes involved in NF-kB signaling pathways, the molecular mechanism of aberrant NF-kB activation in psoriasis remains largely unknown. In this study, we have provided experimental evidence to suggest that the inactivation of PIASy contributes to constitutive NF-kB activation and psoriasis pathogenesis. PIASy-deficient mice developed exacerbated psoriasis-like phenotypes with sustained NF-kB activation in response to topical imiquimod treatment. PIASy deficiency resulted in robust expression of psoriatic cytokines and chemokines from keratinocytes in response to TNFa and IL-17A. Biochemically, we have identified Piasy as a NF-kB associated protein. Piasy protein bound to the NFkB RelA subunit through its C-terminus. Piasy negatively regulated NF-kB activity. Negative regulation of NF-kB was not mediated through its E3 SUMO ligase activity. Piasy inhibited RelA DNA binding in vitro measured by DNA affinity immunoblotting. Piasy protein also bound to the NF-kB binding sites in the CCL20 promoter in vivo as detected by ChIP assay. These lines of evidence suggest that Piasy represses NF-kB activity through inhibiting RelA DNA binding. PIASy is a transcriptional repressor that localizes in the nucleus. Immunostaining analysis of PIASy in psoriatic skin revealed that PIASy cytoplasmic localization was elevated in psoriatic compared to healthy epidermis. Taken together, it suggests that PIASy negatively regulates NF-kB and PIASy inactivation contributes to psoriasis pathogenesis.
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