Intravenous Ig Ameliorates Disease in a Murine Model of Anti-Laminin 332 Mucous Membrane Pemphigoid.

Intravenous Ig (IVIg) is used to treat mucous membrane pemphigoid, although its therapeutic effectivity is not sufficiently supported by randomized controlled clinical trials, and its mode of action is only insufficiently understood. We have examined the effect of IVIg in a mouse model of anti-laminin 332 mucous membrane pemphigoid and found that IVIg ameliorates both cutaneous and mucosal inflammatory lesions. Our investigation into the modes of action of IVIg in mucous membrane pemphigoid indicated effective anti-inflammatory mechanisms beyond the enhanced degradation of IgG mediated through inhibition of the FcRn. Our results suggest that IVIg curbs the activation of neutrophils at several levels. This includes a direct, immediate inhibitory effect on neutrophil activation by immune complexes but not C5a, which blunts the release of ROS and leukotriene B4 from neutrophils. IVIg also suppresses the formation of neutrophil extracellular traps in response to calcium ion ionophore. In vivo treatment with IVIg altered the transcriptome of blood leukocytes and bone marrow neutrophils toward less proinflammatory phenotypes. Collectively, our results support the effectivity of IVIg in the treatment of mucous membrane pemphigoid and indicate that effects on neutrophils at multiple levels may significantly contribute to its therapeutic effects.

Monocyte populations are involved in the pathogenesis of experimental epidermolysis bullosa acquisita.

Monocytes play a significant role in the pathogenesis of most inflammatory diseases, including autoimmune diseases. Herein, different subpopulations of monocytes often play differential, partially antagonistic roles, in the regulation of tissue populations. Pemphigoid diseases constitute a group of autoimmune blistering skin diseases featuring a marked infiltration of the dermis with immune cells, including monocytes. The monocyte subsets infiltrating the skin, however, have largely remained elusive. Monocyte adhesion and recruitment into the inflamed tissues are regulated by chemokine receptors, most prominently by CCR2 and CX3CR1. To delineate the involvement of monocyte populations in autoimmune blistering skin diseases, we spatiotemporally monitored the dynamic spectrum of monocyte populations that infiltrate the inflamed skin using multiphoton intravital imaging and reporter mice for chemokine receptors. Experimental epidermolysis bullosa acquisita (EBA) was induced by injection of anti-murine type VII collagen (amCOLVII) IgG into the Csf1rEGFP-reporter mice, where circulating myeloid cells, such as monocytes and neutrophils, express an EGFP. EGFP+ cells, including neutrophils and monocytes, were present in the skin, immediately after the deposition of the amCOLVII antibody at the dermal-epidermal junction. To investigate the recruitment and involvement of different monocyte-derived cell populations in the disease course further, EBA was induced in CCR2RFP/+-reporter and CX3CR1GFP/+-reporter mice. A comparable distribution of red fluorescent protein (RFP)+ or green fluorescent protein (GFP)+ was found in both diseased mice and their respective controls over time, indicating the similar recruitment of monocytes into the skin following the binding of autoantibodies. Experiments were extended to the CCR2RFP/RFP-deficient and CX3CR1GFP/GFP-deficient mice to determine whether monocyte recruitment and disease severity are compromised in the absence of the receptor. A comparable pattern was seen in the recruitment of monocytes into the skin in both reporter and deficient mice. However, in contrast to similar disease severity between CX3CR1-deficient and reporter mice, CCR2-deficient mice developed significantly less disease than CCR2-reporter mice, as indicated by the percentage of affected area of ears. Collectively, our observations indicate that while CCR2 and CX3CR1 receptors are not involved in the recruitment of monocytes into the skin, CCR2 deficiency is associated with improved disease outcomes in experimental EBA in mice.

C-type lectin receptor expression is a hallmark of neutrophils infiltrating the skin in epidermolysis bullosa acquisita.

Introduction: Inflammatory epidermolysis bullosa acquisita (EBA) is characterized by a neutrophilic response to anti-type VII collagen (COL7) antibodies resulting in the development of skin inflammation and blistering. The antibody transfer model of EBA closely mirrors this EBA phenotype. Methods: To better understand the changes induced in neutrophils upon recruitment from peripheral blood into lesional skin in EBA, we performed single-cell RNA-sequencing of whole blood and skin dissociate to capture minimally perturbed neutrophils and characterize their transcriptome. Results: Through this approach, we identified clear distinctions between circulating activated neutrophils and intradermal neutrophils. Most strikingly, the gene expression of multiple C-type lectin receptors, which have previously been reported to orchestrate host defense against fungi and select bacteria, were markedly dysregulated. After confirming the upregulation of Clec4n, Clec4d, and Clec4e in experimental EBA as well as in lesional skin from patients with inflammatory EBA, we performed functional studies in globally deficient Clec4e-/- and Clec4d-/- mice as well as in neutrophil-specific Clec4n-/- mice. Deficiency in these genes did not reduce disease in the EBA model. Discussion: Collectively, our results suggest that while the upregulation of Clec4n, Clec4d, and Clec4e is a hallmark of activated dermal neutrophil populations, their individual contribution to the pathogenesis of EBA is dispensable.

Dapsone Suppresses Disease in Preclinical Murine Models of Pemphigoid Diseases.

Epidermolysis bullosa acquisita and mucous membrane pemphigoid are autoimmune blistering diseases characterized by mucocutaneous blisters elicited by an autoantibody-mediated immune response against specific proteins of the epidermal basement membrane. The antibiotic dapsone is frequently used to treat both diseases, but its therapeutic effectiveness is uncertain, and its mode of action in these diseases is largely unknown. We evaluated the effect of dapsone in antibody transfer mouse models of epidermolysis bullosa acquisita and mucous membrane pemphigoid, which do not allow the drawing of conclusions on clinical treatment regimens but can be instrumental to partially uncover the mode(s) of action of dapsone in these diseases. Dapsone significantly mitigated inflammation in both models, reducing the recruitment of neutrophils into the skin and disrupting their release of leukotriene B4 (LTB4) and ROS in response to immune complexes. LTB4 has been implicated in numerous diseases, but effective LTB4 inhibitors for clinical use are not available. Our findings indicate that the mode of action of dapsone in these models may be based on the inhibition of LTB4 and ROS release from neutrophils. Moreover, they encourage testing the use of dapsone as an effective, albeit nonspecific, inhibitor of LTB4 biosynthesis in other LTB4-driven diseases.

Immunoglobulin G of systemic sclerosis patients programs a pro-inflammatory and profibrotic phenotype in monocyte-like THP-1 cells.

OBJECTIVES: Functional IgG autoantibodies against diverse G protein-coupled receptors, i.e. antibodies with agonistic or antagonistic activity at these receptors, are abundant in human serum. Their levels are altered in patients with SSc, and autoantibodies against angiotensin II receptor 1 (ATR1) and endothelin receptor A (ETA) have been suggested to drive SSc by inducing the chemokines CXCL8 and CCL18 in the blood. The objective of our study is to profile the effect of IgG in SSc (SSc-IgG) on the production of soluble mediators in monocytic cells. METHODS: Monocyte-like THP-1 cells were stimulated with SSc-IgG and their secretome was analysed. Furthermore, the significance of major pro-inflammatory pathways for the induction of CXCL8 and CCL18 in response to SSc-IgG was assessed by a pharmacological approach. RESULTS: Stimulation with SSc-IgG significantly alters the secretome of THP-1 cells towards a general pro-inflammatory and profibrotic phenotype, which includes an increase of CCL18 and CXCL8. The consequent expression profiles vary depending on the individual donor of the SSc-IgG. CCL18 and CXCL8 expression is thus regulated differentially, with AP-1 driving the induction of both CCL18 and CXCL8 and the TAK/IKK-ß/NF-κB pathway and ERK1/2 driving that of CXCL8. CONCLUSIONS: Our results suggest that SSc-IgG contributes to the generation of the pro-inflammatory/profibrotic tissue milieu characteristic of SSc by its induction of a respective phenotype in monocytes. Furthermore, our results highlight AP-1 as a critical regulator of gene transcription of CCL18 in monocytic cells and as a promising pharmacological therapeutic target for the treatment of SSc.

Cytomegalovirus distribution and evolution in hominines.

Herpesviruses are thought to have evolved in very close association with their hosts. This is notably the case for cytomegaloviruses (CMVs; genus Cytomegalovirus) infecting primates, which exhibit a strong signal of co-divergence with their hosts. Some herpesviruses are however known to have crossed species barriers. Based on a limited sampling of CMV diversity in the hominine (African great ape and human) lineage, we hypothesized that chimpanzees and gorillas might have mutually exchanged CMVs in the past. Here, we performed a comprehensive molecular screening of all 9 African great ape species/subspecies, using 675 fecal samples collected from wild animals. We identified CMVs in eight species/subspecies, notably generating the first CMV sequences from bonobos. We used this extended dataset to test competing hypotheses with various degrees of co-divergence/number of host switches while simultaneously estimating the dates of these events in a Bayesian framework. The model best supported by the data involved the transmission of a gorilla CMV to the panine (chimpanzee and bonobo) lineage and the transmission of a panine CMV to the gorilla lineage prior to the divergence of chimpanzees and bonobos, more than 800,000 years ago. Panine CMVs then co-diverged with their hosts. These results add to a growing body of evidence suggesting that viruses with a double-stranded DNA genome (including other herpesviruses, adenoviruses, and papillomaviruses) often jumped between hominine lineages over the last few million years.

Dual inhibition of complement factor 5 and leukotriene B4 synergistically suppresses murine pemphigoid disease.

The treatment of most autoimmune diseases still relies on systemic immunosuppression and is associated with severe side effects. The development of drugs that more specifically abrogate pathogenic pathways is therefore most desirable. In nature, such specificity is exemplified, e.g., by the soft tick-derived biotherapeutic Coversin, which locally suppresses immune responses by inhibiting complement factor 5 (C5) and leukotriene B4 (LTB4). C5a, a proteolytic fragment of C5, and LTB4 are critical drivers of skin inflammation in pemphigoid diseases (PDs), a group of autoimmune blistering skin diseases. Here, we demonstrate that both Coversin and its mutated form L-Coversin, which inhibits LTB4 only, dose dependently attenuate disease in a model of bullous pemphigoid-like epidermolysis bullosa acquisita (BP-like EBA). Coversin, however, reduces disease more effectively than L-Coversin, indicating that inhibition of C5 and LTB4 synergize in their suppressing effects in this model. Further supporting the therapeutic potential of Coversin in humans, we found that C5a and LTB4 are both present in the blister fluid of patients with BP in quantities inducing the recruitment of granulocytes and that the number of cells expressing their receptors, C5aR1 and BLT1, respectively, is increased in perilesional skin. Collectively, our results highlight Coversin and possibly L-Coversin as potential therapeutics for PDs.

Endosomal Toll-Like Receptors 7 and 9 Cooperate in Detection of Murine Gammaherpesvirus 68 Infection.

Murine gammaherpesvirus 68 (MHV68) is a small-animal model suitable for study of the human pathogens Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. Here, we have characterized the roles of the endosomal Toll-like receptor (TLR) escort protein UNC93B, endosomal TLR7, -9, and -13, and cell surface TLR2 in MHV68 detection. We found that the alpha interferon (IFN-α) response of plasmacytoid dendritic cells (pDC) to MHV68 was reduced in Tlr9-/- cells compared to levels in wild type (WT) cells but not completely lost. Tlr7-/- pDC responded similarly to WT. However, we found that in Unc93b-/- pDC, as well as in Tlr7-/-Tlr9-/- double-knockout pDC, the IFN-α response to MHV68 was completely abolished. Thus, the only pattern recognition receptors contributing to the IFN-α response to MHV68 in pDC are TLR7 and TLR9, but the contribution of TLR7 is masked by the presence of TLR9. To address the role of UNC93B and TLR for MHV68 infection in vivo, we infected mice with MHV68. Lytic replication of MHV68 after intravenous infection was enhanced in the lungs, spleen, and liver of UNC93B-deficient mice, in the spleen of TLR9-deficient mice, and in the liver and spleen of Tlr7-/-Tlr9-/- mice. The absence of TLR2 or TLR13 did not affect lytic viral titers. We then compared reactivation of MHV68 from latently infected WT, Unc93b-/-, Tlr7-/-Tlr9-/-, Tlr7-/-, and Tlr9-/- splenocytes. We observed enhanced reactivation and latent viral loads, particularly from Tlr7-/-Tlr9-/- splenocytes compared to levels in the WT. Our data show that UNC93B-dependent TLR7 and TLR9 cooperate in and contribute to detection and control of MHV68 infection.IMPORTANCE The two human gammaherpesviruses, Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV), can cause aggressive forms of cancer. These herpesviruses are strictly host specific, and therefore the homolog murine gammaherpesvirus 68 (MHV68) is a widely used model to obtain in vivo insights into the interaction between these two gammaherpesviruses and their host. Like EBV and KSHV, MHV68 establishes lifelong latency in B cells. The innate immune system serves as one of the first lines of host defense, with pattern recognition receptors such as the Toll-like receptors playing a crucial role in mounting a potent antiviral immune response to various pathogens. Here, we shed light on a yet unanticipated role of Toll-like receptor 7 in the recognition of MHV68 in a subset of immune cells called plasmacytoid dendritic cells, as well as on the control of this virus in its host.

Cytomegaloviruses in a Community of Wild Nonhuman Primates in Taï National Park, Côte D'Ivoire.

Cytomegaloviruses (CMVs) are known to infect many mammals, including a number of nonhuman primates (NHPs). However, most data available arose from studies led on captive individuals and little is known about CMV diversity in wild NHPs. Here, we analyzed a community of wild nonhuman primates (seven species) in Taï National Park (TNP), Côte d'Ivoire, with two PCR systems targeting betaherpesviruses. CMV DNA was detected in 17/87 primates (4/7 species). Six novel CMVs were identified in sooty mangabeys, Campbell's monkeys and Diana monkeys, respectively. In 3/17 positive individuals (from three NHP species), different CMVs were co-detected. A major part of the glycoprotein B coding sequences of the novel viruses was amplified and sequenced, and phylogenetic analyses were performed that included three previously discovered CMVs of western red colobus from TNP and published CMVs from other NHP species and geographic locations. We find that, despite this locally intensified sampling, NHP CMVs from TNP are completely host-specific, pinpointing the absence or rarity of cross-species transmission. We also show that on longer timescales the evolution of CMVs is characterized by frequent co-divergence with their hosts, although other processes, including lineage duplication and host switching, also have to be invoked to fully explain their evolutionary relationships.

Absence of frequent herpesvirus transmission in a nonhuman primate predator-prey system in the wild.

Emergence of viruses into the human population by transmission from nonhuman primates (NHPs) represents a serious potential threat to human health that is primarily associated with the increased bushmeat trade. Transmission of RNA viruses across primate species appears to be relatively frequent. In contrast, DNA viruses appear to be largely host specific, suggesting low transmission potential. Herein, we use a primate predator-prey system to study the risk of herpesvirus transmission between different primate species in the wild. The system was comprised of western chimpanzees (Pan troglodytes verus) and their primary (western red colobus, Piliocolobus badius badius) and secondary (black-and-white colobus, Colobus polykomos) prey monkey species. NHP species were frequently observed to be coinfected with multiple beta- and gammaherpesviruses (including new cytomegalo- and rhadinoviruses). However, despite frequent exposure of chimpanzees to blood, organs, and bones of their herpesvirus-infected monkey prey, there was no evidence for cross-species herpesvirus transmission. These findings suggest that interspecies transmission of NHP beta- and gammaherpesviruses is, at most, a rare event in the wild.