Cysteine Oxidation in Human Galectin-1 Occurs Sequentially via a Folded Intermediate to a Fully Oxidized Unfolded Form.

Galectins are multifunctional effectors in cellular homeostasis and dysregulation. Oxidation of human galectin-1 (Gal-1) with its six sulfhydryls produces a disulfide-bridged oxidized form that lacks normal lectin activity yet gains new glycan-independent functionality. Nevertheless, the mechanistic details as to how Gal-1 oxidation occurs remain unclear. Here, we used 15N and 13C HSQC NMR spectroscopy to gain structural insight into the CuSO4-mediated path of Gal-1 oxidation and identified a minimum two-stage conversion process. During the first phase, disulfide bridges form slowly between C16-C88 and/or C42-C66 to produce a partially oxidized, conformationally flexible intermediate that retains the ability to bind lactose. Site-directed mutagenesis of C16 to S16 impedes the onset of this overall slow process. During the second phase, increased motional dynamics of the intermediate enable the relatively distant C2 and C130 residues to form the third and final disulfide bond, leading to an unfolded state and consequent dimer dissociation. This fully oxidized end state loses the ability to bind lactose, as shown by the hemagglutination assay. Consistent with this model, we observed that the Gal-1 C2S mutant maintains intermediate-state structural features with a free sulfhydryl group at C130. Incubation with dithiothreitol reduces all disulfide bonds and allows the lectin to revert to its native state. Thus, the sequential, non-random formation of three disulfide bridges in Gal-1 in an oxidative environment acts as a molecular switch for fundamental changes to its functionality. These data inspire detailed bioactivity analysis of the structurally defined oxidized intermediate in, e.g., acute and chronic inflammation.

Complement Membrane Attack Complexes Disrupt Proteostasis to Function as Intracellular Alarmins.

Internalized pools of membrane attack complexes (MACs) promote NF-kB and dysregulated tissue inflammation. Here, we show that C9, a MAC-associated protein, promotes loss of proteostasis to become intrinsically immunogenic. Surface-bound C9 is internalized into Rab5 + endosomes whose intraluminal acidification promotes C9 aggregates. A region within the MACPF/CDC domain of C9 stimulates aggrephagy to induce NF-kB, inflammatory genes, and EC activation. This process requires ZFYVE21, a Rab5 effector, which links LC3A/B on aggresome membranes to RNF34-P62 complexes to mediate C9 aggrephagy. C9 aggregates form in human tissues, C9-associated signaling responses occur in three mouse models, and ZFYVE21 stabilizes RNF34 to promote C9 aggrephagy in vivo. Gene-deficient mice lacking ZFYVE21 in ECs showed reduced MAC-induced tissue injury in a skin model of chronic rejection. While classically defined as cytotoxic effectors, MACs may impair proteostasis, forming aggregates that behave as intracellular alarmins.

Cutaneous Wound Healing and the Effects of Cannabidiol.

Cutaneous wounds, both acute and chronic, begin with loss of the integrity, and thus barrier function, of the skin. Surgery and trauma produce acute wounds. There are 22 million surgical procedures per year in the United States alone, based on data from the American College of Surgeons, resulting in a prevalence of 6.67%. Acute traumatic wounds requiring repair total 8 million per year, 2.42% or 24.2 per 1000. The cost of wound care is increasing; it approached USD 100 billion for just Medicare in 2018. This burden for wound care will continue to rise with population aging, the increase in metabolic syndrome, and more elective surgeries. To heal a wound, an orchestrated, evolutionarily conserved, and complex series of events involving cellular and molecular agents at the local and systemic levels are necessary. The principal factors of this important function include elements from the neurological, cardiovascular, immune, nutritional, and endocrine systems. The objectives of this review are to provide clinicians engaged in wound care and basic science researchers interested in wound healing with an updated synopsis from recent publications. We also present data from our primary investigations, testing the hypothesis that cannabidiol can alter cutaneous wound healing and documenting their effects in wild type (C57/BL6) and db/db mice (Type 2 Diabetes Mellitus, T2DM). The focus is on the potential roles of the endocannabinoid system, cannabidiol, and the important immune-regulatory wound cytokine IL-33, a member of the IL-1 family, and connective tissue growth factor, CTGF, due to their roles in both normal and abnormal wound healing. We found an initial delay in the rate of wound closure in B6 mice with CBD, but this difference disappeared with time. CBD decreased IL-33 + cells in B6 by 70% while nearly increasing CTGF + cells in db/db mice by two folds from 18.6% to 38.8% (p < 0.05) using a dorsal wound model. We review the current literature on normal and abnormal wound healing, and document effects of CBD in B6 and db/db dorsal cutaneous wounds. CBD may have some beneficial effects in diabetic wounds. We applied 6-mm circular punch to create standard size full-thickness dorsal wounds in B6 and db/db mice. The experimental group received CBD while the control group got only vehicle. The outcome measures were rate of wound closure, wound cells expressing IL-33 and CTGF, and ILC profiles. In B6, the initial rate of wound closure was slower but there was no delay in the time to final closure, and cells expressing IL-33 was significantly reduced. CTGF + cells were higher in db/bd wounds treated with CBD. These data support the potential use of CBD to improve diabetic cutaneous wound healing.

Receptor for Advanced Glycation End Product (RAGE) Modulates Inflammation During Feeding of the Hard Tick, Haemaphysalis longicornis in Mice.

Ticks are notorious blood-sucking ectoparasites that affect both humans and animals. They serve as a unique vector of various deadly diseases. Here, we have shown the roles of the receptor for advanced glycation end products (RAGE) during repeated infestations by the tick Haemaphysalis longicornis using RAGE-/- mice. In primary infestation, a large blood pool developed, which was flooded with numerous RBCs, especially during the rapid feeding phase of the tick both in wild-type (wt) and RAGE-/- mice. Very few inflammatory cells were detected around the zones of haemorrhage in the primary infestations. However, the number of inflammatory cells gradually increased in the subsequent tick infestations, and during the third infestations, the number of inflammatory cells reached to the highest level (350.3 ± 16.8 cells/focus). The site of attachment was totally occupied by the inflammatory cells in wt mice, whereas very few cells were detected at the ticks' biting sites in RAGE-/- mice. RAGE was highly expressed during the third infestation in wt mice. In the third infestation, infiltration of CD44+ lymphocytes, eosinophils and expression of S100A8 and S100B significantly increased at the biting sites of ticks in wt, but not in RAGE-/- mice. In addition, peripheral eosinophil counts significantly increased in wt but not in RAGE-/- mice. Taken together, our study revealed that RAGE-mediated inflammation and eosinophils played crucial roles in the tick-induced inflammatory reactions.

Immune Response Dynamics and Biomarkers in COVID-19 Patients.

BACKGROUND: The immune response dynamics in COVID-19 patients remain a subject of intense investigation due to their implications for disease severity and treatment outcomes. We examined changes in leukocyte levels, eosinophil activity, and cytokine profiles in patients hospitalized with COVID-19. METHODS: Serum samples were collected within the first 10 days of hospitalization/confirmed infection and analyzed for eosinophil granule proteins (EGP) and cytokines. Information from medical records including comorbidities, clinical symptoms, medications, and complete blood counts were collected at the time of admission, during hospitalization and at follow up approximately 3 months later. RESULTS: Serum levels of eotaxin, type 1 and type 2 cytokines, and alarmin cytokines were elevated in COVID-19 patients, highlighting the heightened immune response (p < 0.05). However, COVID-19 patients exhibited lower levels of eosinophils and eosinophil degranulation products compared to hospitalized controls (p < 0.05). Leukocyte counts increased consistently from admission to follow-up, indicative of recovery. CONCLUSION: Attenuated eosinophil activity alongside elevated chemokine and cytokine levels during active infection, highlights the complex interplay of immune mediators in the pathogenesis COVID-19 and underscores the need for further investigation into immune biomarkers and treatment strategies.

Processing of angiocrine alarmin IL-1α in endothelial cells promotes lung and liver fibrosis.

BACKGROUND: Fibrosis results from excessive scar formation after tissue injury. Injured cells release alarmins such as interleukin 1 (IL-1) α and ß as primary mediators initiating tissue repair. However, how alarmins from different cell types differentially regulate fibrosis remains to be explored. METHODS: Here, we used tissue specific knockout strategy to illustrate a unique contribution of endothelial cell-derived IL-1α to lung and liver fibrosis. The two fibrotic animal model triggered by bleomycin and CCl4 were used to study the effects of endothelial paracrine/angiocrine IL-1α in fibrotic progression. Human umbilical vein endothelial cells (HUVEC) were performed to explore the production of angiocrine IL-1α at both transcriptional and post-transcriptional levels in vitro. RESULTS: We found that endothelial paracrine/angiocrine IL-1α primarily promotes lung and liver fibrosis during the early phase of organ repair. By contrast, myeloid cell-specific ablation of IL-1α in mice resulted in little influence on fibrosis, suggesting the specific pro-fibrotic role of IL-1α from endothelial cell but not macrophage. In vitro study revealed a coordinated regulation of IL-1α production in human primary endothelial cells at both transcriptional and post-transcriptional levels. Specifically, the transcription of IL-1α is regulated by RIPK1, and after caspase-8 (CASP8) cleaves the precursor form of IL-1α, its secretion is triggered by ion channel Pannexin 1 upon CASP8 cleavage. CONCLUSIONS: Endothelial cell-produced IL-1α plays a unique role in promoting organ fibrosis. Furthermore, the release of this angiocrine alarmin relies on a unique molecular mechanism involving RIPK1, CASP8, and ion channel Pannexin 1.

Alarmins in cutaneous malignant melanoma: An updated overview of emerging evidence on their pathogenetic, diagnostic, prognostic, and therapeutic role.

Malignant cutaneous melanoma is the leading cause of death for skin cancer to date, with globally increasing incidence rates. In this epidemiological scenario, international scientific research is exerting efforts to identify new clinical strategies aimed at the prognostic amelioration of the disease. Very promising and groundbreaking in this context is the scientific interest related to alarmins and their pioneering utility in the setting of the pathogenetic understanding, diagnosis, prognosis, and therapy for malignant cutaneous melanoma. However, the scientific investigations on this matter should not overlook their still well-presented dual and contradictory role. The aim of our critical analysis is to provide an up-to-date overview of the emerging evidence concerning the dichotomous role of alarmins in the aforementioned clinical settings. Our literature revision was based on the extensive body of both preclinical and clinical findings published on the PubMed database over the past 5 years. In addition to this, we offer a special focus on potentially revolutionary new therapeutic frontiers, which, on the strength of their earliest successes in other clinical areas, could inaugurate a new era of personalized and precision medicine in the field of dermato-oncology.

Tezepelumab: patient selection and place in therapy in severe asthma.

Asthma is a disease characterised by heterogeneous and multifaceted airway inflammation. Despite the availability of effective treatments, a substantial percentage of patients with the type 2 (T2)-high, but mainly the T2-low, phenotype complain of persistent symptoms, airflow limitation, and poor response to treatments. Currently available biologicals target T2 cytokines, but no monoclonal antibodies or other specific therapeutic options are available for non-T2 asthma. However, targeted therapy against alarmins is radically changing this perspective. The development of alarmin-targeted therapies, of which tezepelumab (TZP) is the first example, may offer broad action on inflammatory pathways as well as an enhanced therapeutic effect on epithelial dysfunction. In this regard, TZP demonstrated positive results not only in patients with severe T2 asthma but also those with non-allergic, non-eosinophilic disease. Therefore, it is necessary to identify clinical features of patients who can benefit from an upstream targeted therapy such as anti-thymic stromal lymphopoietin. The aims of this narrative review are to understand the role of alarmins in asthma pathogenesis and epithelial dysfunction, examine the rationale underlying the indication of TZP treatment in severe asthma, summarise the results of clinical studies, and recognise the specific characteristics of patients potentially eligible for TZP treatment.

Bactericidal/permeability-increasing protein instructs dendritic cells to elicit Th22 cell response.

Neutrophil-derived bactericidal/permeability-increasing protein (BPI) is known for its bactericidal activity against gram-negative bacteria and neutralization of lipopolysaccharide. Here, we define BPI as a potent activator of murine dendritic cells (DCs). As shown in GM-CSF-cultured, bone-marrow-derived cells (BMDCs), BPI induces a distinct stimulation profile including IL-2, IL-6, and tumor necrosis factor expression. Conventional DCs also respond to BPI, while M-CSF-cultivated or peritoneal lavage macrophages do not. Subsequent to BPI stimulation of BMDCs, CD4+ T cells predominantly secrete IL-22 and, when naive, preferentially differentiate into T helper 22 (Th22) cells. Congruent with the tissue-protective properties of IL-22 and along with impaired IL-22 induction, disease severity is significantly increased during dextran sodium sulfate-induced colitis in BPI-deficient mice. Importantly, physiological diversification of intestinal microbiota fosters BPI-dependent IL-22 induction in CD4+ T cells derived from mesenteric lymph nodes. In conclusion, BPI is a potent activator of DCs and consecutive Th22 cell differentiation with substantial relevance in intestinal homeostasis.

S100A4 a classical DAMP as a therapeutic target in fibrosis.

Fibrosis regardless of aetiology is characterised by persistently activated myofibroblasts that are contractile and secrete excessive amounts of extracellular matrix molecules that leads to loss of organ function. Damage-Associated Molecular Patterns (DAMPs) are endogenous host-derived molecules that are released from cells dying or under stress that can be triggered by a variety of insults, either chemical or physical, leading to an inflammatory response. Among these DAMPs is S100A4, part of the S100 family of calcium binding proteins that participate in a variety of cellular processes. S100A4 was first described in context of cancer as a pro-metastatic factor. It is now appreciated that aside from its role in cancer promotion, S100A4 is intimately involved in tissue fibrosis. The extracellular form of S100A4 exerts its effects through multiple receptors including Toll-Like Receptor 4 and RAGE to evoke signalling cascades involving downstream mediators facilitating extracellular matrix deposition and myofibroblast generation and can play a role in persistent activation of myofibroblasts. S100A4 may be best understood as an amplifier of inflammatory and fibrotic processes. S100A4 appears critical in systemic sclerosis pathogenesis and blocking the extracellular form of S100A4 in vivo in various animal models of disease mitigates fibrosis and may even reverse established disease. This review appraises S100A4's position as a DAMP and its role in fibrotic conditions and highlight therapeutically targeting this protein to halt fibrosis, suggesting that it is a tractable target.

Blockade of IL-6R prevents preterm birth and adverse neonatal outcomes.

BACKGROUND: Preterm birth preceded by spontaneous preterm labour often occurs in the clinical setting of sterile intra-amniotic inflammation (SIAI), a condition that currently lacks treatment. METHODS: Proteomic and scRNA-seq human data were analysed to evaluate the role of IL-6 and IL-1α in SIAI. A C57BL/6 murine model of SIAI-induced preterm birth was developed by the ultrasound-guided intra-amniotic injection of IL-1α. The blockade of IL-6R by using an aIL-6R was tested as prenatal treatment for preterm birth and adverse neonatal outcomes. QUEST-MRI evaluated brain oxidative stress in utero. Targeted transcriptomic profiling assessed maternal, foetal, and neonatal inflammation. Neonatal biometrics and neurodevelopment were tested. The neonatal gut immune-microbiome was evaluated using metagenomic sequencing and immunophenotyping. FINDINGS: IL-6 plays a critical role in the human intra-amniotic inflammatory response, which is associated with elevated concentrations of the alarmin IL-1α. Intra-amniotic injection of IL-1α resembles SIAI, inducing preterm birth (7% vs. 50%, p = 0.03, Fisher's exact test) and neonatal mortality (18% vs. 56%, p = 0.02, Mann-Whitney U-test). QUEST-MRI revealed no foetal brain oxidative stress upon in utero IL-1α exposure (p > 0.05, mixed linear model). Prenatal treatment with aIL-6R abrogated IL-1α-induced preterm birth (50% vs. 7%, p = 0.03, Fisher's exact test) by dampening inflammatory processes associated with the common pathway of labour. Importantly, aIL-6R reduces neonatal mortality (56% vs. 22%, p = 0.03, Mann-Whitney U-test) by crossing from the mother to the amniotic cavity, dampening foetal organ inflammation and improving growth. Beneficial effects of prenatal IL-6R blockade carried over to neonatal life, improving survival, growth, neurodevelopment, and gut immune homeostasis. INTERPRETATION: IL-6R blockade can serve as a strategy to treat SIAI, preventing preterm birth and adverse neonatal outcomes. FUNDING: NICHD/NIH/DHHS, Contract HHSN275201300006C. WSU Perinatal Initiative in Maternal, Perinatal and Child Health.

S100A alarmins and thymic stromal lymphopoietin (TSLP) regulation in severe asthma following bronchial thermoplasty.

RATIONALE: Severe asthma affects a small proportion of asthmatics but represents a significant healthcare challenge. Bronchial thermoplasty (BT) is an interventional treatment approach preconized for uncontrolled severe asthma after considering biologics therapy. It was showed that BT long-lastingly improves asthma control. These improvements seem to be related to the ability of BT to reduce airway smooth muscle remodeling, reduce the number of nerve fibers and to modulate bronchial epithelium integrity and behavior. Current evidence suggest that BT downregulates epithelial mucins expression, cytokine production and metabolic profile. Despite these observations, biological mechanisms explaining asthma control improvement post-BT are still not well understood. OBJECTIVES: To assess whether BT affects gene signatures in bronchial epithelial cells (BECs). METHODS: In this study we evaluated the transcriptome of cultured bronchial epithelial cells (BECs) of severe asthmatics obtained pre- and post-BT treatment using microarrays. We further validated gene and protein expressions in BECs and in bronchial biopsies with immunohistochemistry pre- and post-BT treatment. MEASUREMENTS AND MAIN RESULTS: Transcriptomics analysis revealed that a large portion of differentially expressed genes (DEG) was involved in anti-viral response, anti-microbial response and pathogen induced cytokine storm signaling pathway. S100A gene family stood out as five members of this family where consistently downregulated post-BT. Further validation revealed that S100A7, S100A8, S100A9 and their receptor (RAGE, TLR4, CD36) expressions were highly enriched in severe asthmatic BECs. Further, these S100A family members were downregulated at the gene and protein levels in BECs and in bronchial biopsies of severe asthmatics post-BT. TLR4 and CD36 protein expression were also reduced in BECs post-BT. Thymic stromal lymphopoietin (TSLP) and human ß-defensin 2 (hBD2) were significantly decreased while no significant change was observed in IL-25 and IL-33. CONCLUSIONS: These data suggest that BT might improve asthma control by downregulating epithelial derived S100A family expression and related downstream signaling pathways.

Serum alarmins and the risk of incident interstitial lung disease in rheumatoid arthritis.

OBJECTIVES: To quantify associations of serum alarmins with risk of rheumatoid arthritis-associated interstitial lung disease (RA-ILD). METHODS: Using serum collected at enrolment, three alarmins (interleukin [IL]-33, thymic stromal lymphopoietin [TSLP], and IL-25) were measured in a multicentre prospective RA cohort. ILD was classified using systematic medical record review. Cross-sectional associations of log-transformed (IL-33, TSLP) or quartile (IL-25) values with RA-ILD at enrolment (prevalent RA-ILD) were examined using logistic regression, while associations with incident RA-ILD developing after enrolment were examined using Cox proportional hazards. Covariates in multivariate models included age, sex, race, smoking status, RA disease activity score, and anti-cyclic citrullinated antibody positivity. RESULTS: Of 2,835 study participants, 115 participants (4.1%) had prevalent RA-ILD at baseline and an additional 146 (5.1%) developed incident ILD. There were no associations between serum alarmin concentrations and prevalent ILD in unadjusted or adjusted logistic regression models. In contrast, there was a significant inverse association between IL-33 concentration and the risk of developing incident RA-ILD in unadjusted (HR 0.73 per log-fold increase; 95% CI 0.57-0.95; p= 0.018) and adjusted (HR 0.77; 95% CI 0.59-1.00, p= 0.047) models. No significant associations of TSLP or IL-25 with incident ILD were observed. CONCLUSIONS: In this study, we observed a significant inverse association between serum IL-33 concentration and the risk of developing incident RA-ILD, but no associations with prevalent ILD. Additional investigation is required to better understand the mechanisms driving this relationship and how serum alarmin IL-33 assessment might contribute to clinical risk stratification in patients with RA.

Efficacy and Potential Positioning of Tezepelumab in the Treatment of Severe Asthma.

The excellent results for monoclonal antibodies in the treatment of severe uncontrolled asthma (SUCA) represent a milestone in current treatment of asthmatic disorders. Remaining, however, are several subsidiary areas for improvement in which new biologics are expected to make a decisive contribution. These biologics include tezepelumab, a monoclonal antibody that blocks thymic stromal lymphopoietin (TSLP). TSLP is an epithelial-release cytokine (alarmin) that plays a key role in initiating both the innate (group 2 innate lymphoid cell (ILC) pathway) and the acquired (T helper 2 (Th2) pathway) immune responses by activating the type 2 (T2) asthma inflammatory pathway through both. It is also thought that it may additionally intervene in the neutrophilic non-T2 inflammatory pathway (via interaction with ILC3 and interleukin-17). Six clinical trials that included 2187 patients with uncontrolled asthma, with 2 or more exacerbations in the previous year, on medium/high-dose inhaled corticosteroids and at least 1 other controller, have demonstrated - irrespective of T2 endotype (and possibly also non-T2 endotype) - the efficacy and safety of tezepelumab, as it significantly reduces exacerbations (61.7%-66%) and bronchial hyperresponsiveness, and improves lung function, disease control, and quality of life. Tezepelumab could be indicated for the treatment of patients with, independently of the T2 phenotype (eosinophilic and non-eosinophilic), and may even be the only biologic available for treatment of non-T2 SUCA.

Los excelentes resultados de los anticuerpos monoclonales en el tratamiento del asma grave no controlada (AGNC) constituyen un hito en el tratamiento actual de los trastornos asmáticos. Sin embargo, aún quedan varios aspectos complementarios susceptibles de mejorar para los que se esperan contribuciones decisivas de los nuevos biofármacos, entre los cuales se encuentra el tezepelumab, un anticuerpo monoclonal que bloquea la linfopoyetina estromal tímica (TSLP). La TSLP es una citocina de liberación epitelial (alarmina) que desempeña una función clave en el inicio de las respuestas inmunitarias tanto innata (vía de las células linfocíticas innatas [ILC] del grupo 2) como adaptativa (vía de los linfocitos T cooperadores 2 [Th2]), activando la vía inflamatoria del asma del tipo 2 (T2) mediante ambas. También se cree que puede intervenir en la vía inflamatoria neutrofílica con T2 baja (mediante la interacción con los ILC3 y la interleucina 17). En seis ensayos clínicos que incluyeron a 2.187 pacientes con asma no controlada, dos o más exacerbaciones en el año anterior, a tratamiento con corticosteroides inhalados en dosis medias o altas y con un mínimo de un tratamiento preventivo adicional, se ha demostrado la eficacia y seguridad del tezepelumab sin importar el endotipo T2 (y posiblemente tampoco el endotipo no T2), ya que reduce significativamente las exacerbaciones (61,7-66%) y la hiperreactividad bronquial y mejora la función pulmonar, el control de la enfermedad y la calidad de vida. El tezepelumab puede estar indicado para tratar a pacientes con asma grave, independientemente del fenotipo T2 (eosinofílico y no eosinofílico), y tal vez sea incluso el único biofármaco existente para el tratamiento del AGNC no T2.

Development of a novel modified vaccine (TheraVacM) for curative treatment of mouse solid tumors.

Monotherapy with immune checkpoint blockade (ICB) antibodies (anti-CTLA4 and anti-PD1/PDL-1) is only effective for 20% to 30% of patients with certain cancers. Patients with cancers harboring few effector T cells (Teffs) are insensitive to ICB therapy. The lack of tumor-specific Teffs is predominantly caused by the paralysis of tumor-infiltrating dendritic cells (TiDCs) resulting from immunosuppression in the tumor microenvironment. We have identified a potent combination of high mobility group nucleosome binding domain 1 (HMGN1, N1) and fibroblast stimulating lipopeptide-1 (FSL-1) that can synergistically trigger maturation of both mouse and human DCs. Accordingly, we designed a combinational anti-cancer immunotherapy with two arms: an immune-activating arm consisting of N1 and FSL-1 to stimulate the generation of Teffs by triggering full maturation of TiDCs, and an ICB arm using anti-PDL-1 or anti-CTLA4 to prevent Teffs from being silenced in the tumor tissue. This combinational immunotherapeutic vaccination regimen dubbed modified TheraVac (TheraVacM) has proved particularly effective as it cured 100% of mice bearing established ectopic CT26 colon and RENCA kidney tumors. The resultant tumor-free mice were resistant to subsequent re-challenge with the same tumors, indicating the generation of long-term tumor specific protective immunity. Since the immune-activating arm also induces full maturation of human DCs, and anti-PDL-1 or anti-CTLA4 have been FDA-approved, this combinational immunotherapy has the potential to be an effective clinical therapy for patients with solid tumors.

A positive feedback cycle between the alarmin S100A8/A9 and NLRP3 inflammasome-GSDMD signalling reinforces the innate immune response in Candida albicans keratitis.

OBJECTIVE: Fungal keratitis is a severe sight-threatening ocular infection, without effective treatment strategies available now. Calprotectin S100A8/A9 has recently attracted great attention as a critical alarmin modulating the innate immune response against microbial challenges. However, the unique role of S100A8/A9 in fungal keratitis is poorly understood. METHODS: Experimental fungal keratitis was established in wild-type and gene knockout (TLR4-/- and GSDMD-/-) mice by infecting mouse corneas with Candida albicans. The degree of mouse cornea injuries was evaluated by clinical scoring. To interrogate the molecular mechanism in vitro, macrophage RAW264.7 cell line was challenged with Candida albicans or recombinant S100A8/A9 protein. Label-free quantitative proteomics, quantitative real-time PCR, Western blotting, and immunohistochemistry were conducted in this research. RESULTS: Herein, we characterized the proteome of mouse corneas infected with Candida albicans and found that S100A8/A9 was robustly expressed at the early stage of the disease. S100A8/A9 significantly enhanced disease progression by promoting NLRP3 inflammasome activation and Caspase-1 maturation, accompanied by increased accumulation of macrophages in infected corneas. In response to Candida albicans infection, toll-like receptor 4 (TLR4) sensed extracellular S100A8/A9 and acted as a bridge between S100A8/A9 and NLRP3 inflammasome activation in mouse corneas. Furthermore, the deletion of TLR4 resulted in noticeable improvement in fungal keratitis. Remarkably, NLRP3/GSDMD-mediated macrophage pyroptosis in turn facilitates S100A8/A9 secretion during Candida albicans keratitis, thus forming a positive feedback cycle that amplifies the proinflammatory response in corneas. CONCLUSIONS: The present study is the first to reveal the critical roles of the alarmin S100A8/A9 in the immunopathology of Candida albicans keratitis, highlighting a promising approach for therapeutic intervention in the future.

The multifaceted nature of IL-10: regulation, role in immunological homeostasis and its relevance to cancer, COVID-19 and post-COVID conditions.

Interleukin-10 (IL-10) is a pleiotropic cytokine that has a fundamental role in modulating inflammation and in maintaining cell homeostasis. It primarily acts as an anti-inflammatory cytokine, protecting the body from an uncontrolled immune response, mostly through the Jak1/Tyk2 and STAT3 signaling pathway. On the other hand, IL-10 can also have immunostimulating functions under certain conditions. Given the pivotal role of IL-10 in immune modulation, this cytokine could have relevant implications in pathologies characterized by hyperinflammatory state, such as cancer, or infectious diseases as in the case of COVID-19 and Post-COVID-19 syndrome. Recent evidence proposed IL-10 as a predictor of severity and mortality for patients with acute or post-acute SARS-CoV-2 infection. In this context, IL-10 can act as an endogenous danger signal, released by tissues undergoing damage in an attempt to protect the organism from harmful hyperinflammation. Pharmacological strategies aimed to potentiate or restore IL-10 immunomodulatory action may represent novel promising avenues to counteract cytokine storm arising from hyperinflammation and effectively mitigate severe complications. Natural bioactive compounds, derived from terrestrial or marine photosynthetic organisms and able to increase IL-10 expression, could represent a useful prevention strategy to curb inflammation through IL-10 elevation and will be discussed here. However, the multifaceted nature of IL-10 has to be taken into account in the attempts to modulate its levels.

The axis of complement C1 and nucleolus in antinuclear autoimmunity.

Antinuclear autoantibodies (ANA) are heterogeneous self-reactive antibodies that target the chromatin network, the speckled, the nucleoli, and other nuclear regions. The immunological aberration for ANA production remains partially understood, but ANA are known to be pathogenic, especially, in systemic lupus erythematosus (SLE). Most SLE patients exhibit a highly polygenic disease involving multiple organs, but in rare complement C1q, C1r, or C1s deficiencies, the disease can become largely monogenic. Increasing evidence point to intrinsic autoimmunogenicity of the nuclei. Necrotic cells release fragmented chromatins as nucleosomes and the alarmin HMGB1 is associated with the nucleosomes to activate TLRs and confer anti-chromatin autoimmunogenecity. In speckled regions, the major ANA targets Sm/RNP and SSA/Ro contain snRNAs that confer autoimmunogenecity to Sm/RNP and SSA/Ro antigens. Recently, three GAR/RGG-containing alarmins have been identified in the nucleolus that helps explain its high autoimmunogenicity. Interestingly, C1q binds to the nucleoli exposed by necrotic cells to cause protease C1r and C1s activation. C1s cleaves HMGB1 to inactive its alarmin activity. C1 proteases also degrade many nucleolar autoantigens including nucleolin, a major GAR/RGG-containing autoantigen and alarmin. It appears that the different nuclear regions are intrinsically autoimmunogenic by containing autoantigens and alarmins. However, the extracellular complement C1 complex function to dampen nuclear autoimmunogenecity by degrading these nuclear proteins.

Elevated interleukin-11 in systemic sclerosis and role in disease pathogenesis.

Systemic sclerosis (SSc) is an autoimmune connective tissue disease in which there is elevated inflammation, aberrant cytokine expression, and subsequent fibrosis. Interleukin-11 (IL-11) is a recently described profibrotic cytokine that can mediate fibrosis in the heart, lungs, and skin and is upregulated by transforming Growth Factor-ß (TGF-ß1). The objective of this study was to quantify the serum levels of IL-11 in early diffuse SSc patients. Also, if IL-11 could regulate the alarmin IL-33 in dermal fibroblasts was quantified. Early diffuse SSc patient sera was isolated and IL-11 was quantified by specific commercial ELISA compared to healthy control (n = 17). Healthy dermal fibroblasts were cultured in vitro and then serum starved and incubated with or without recombinant IL-11. At specific early and late time points the supernatant was quantified for the alarmin IL-33 by specific ELISA. In early diffuse SSc patients it was demonstrated that they have elevated IL-11 in their sera. In a subgroup of SSc patients with interstitial lung disease (ILD) this elevation was particularly pronounced compared to those devoid of fibrotic lung disease. In vitro incubation of healthy dermal fibroblasts led to a significant induction of IL-33 cytokine release into the cell media. IL-11 is a profibrotic cytokine that is elevated in early diffuse SSc and is particularly elevated in those with ILD. This suggests that IL-11 could be a possible biomarker of ILD in SSc. It was also found that IL-11 led to release of the cytokine alarmin IL-33 in fibroblasts at earlier time points but not late time points, suggesting early stimulation elicits an inflammatory response in the local microenvironment but prolonged stimulation leads to fibrosis.