Oxylipins and metabolites from pyroptotic cells act as promoters of tissue repair.

Pyroptosis is a lytic cell death mode that helps limit the spread of infections and is also linked to pathology in sterile inflammatory diseases and autoimmune diseases1-4. During pyroptosis, inflammasome activation and the engagement of caspase-1 lead to cell death, along with the maturation and secretion of the inflammatory cytokine interleukin-1ß (IL-1ß). The dominant effect of IL-1ß in promoting tissue inflammation has clouded the potential influence of other factors released from pyroptotic cells. Here, using a system in which macrophages are induced to undergo pyroptosis without IL-1ß or IL-1α release (denoted Pyro-1), we identify unexpected beneficial effects of the Pyro-1 secretome. First, we noted that the Pyro-1 supernatants upregulated gene signatures linked to migration, cellular proliferation and wound healing. Consistent with this gene signature, Pyro-1 supernatants boosted migration of primary fibroblasts and macrophages, and promoted faster wound closure in vitro and improved tissue repair in vivo. In mechanistic studies, lipidomics and metabolomics of the Pyro-1 supernatants identified the presence of both oxylipins and metabolites, linking them to pro-wound-healing effects. Focusing specifically on the oxylipin prostaglandin E2 (PGE2), we find that its synthesis is induced de novo during pyroptosis, downstream of caspase-1 activation and cyclooxygenase-2 activity; further, PGE2 synthesis occurs late in pyroptosis, with its release dependent on gasdermin D pores opened during pyroptosis. As for the pyroptotic metabolites, they link to immune cell infiltration into the wounds, and polarization to CD301+ macrophages. Collectively, these data advance the concept that the pyroptotic secretome possesses oxylipins and metabolites with tissue repair properties that may be harnessed therapeutically.

Xanthogranulomatous inflammation of the female genital tract: An 11-year single institutional study of 40 cases.

BACKGROUND AND OBJECTIVES: Xanthogranulomatous inflammation (XGI) is a rare form of chronic inflammation that affects the female genital tract (FGT). The absence of a standard lexicon in the literature has contributed to the relative obscurity of this condition. We attempt to study this lesion with its various clinicopathological associations. METHODOLOGY: We conducted an 11-year retrospective study of cases diagnosed with XGI of the FGT, analyzing relevant clinical and pathological parameters. RESULTS: Our study identified 40 cases reported as XGI. The mean age was 43.8 (+/- 11.8 SD) years. The most common clinical presentation was abdominal pain (27.5 %). Abdominal mass was seen in 37.5 % of which 22.5 % was primarily attributed to XGI and was not associated with malignancies. The most common site involved was adnexa(87.5 %), with rare involvement of myometrium(7.5 %) and endometrium(5 %). Adnexal involvement was either as tubo-ovarian masses or isolated ovary/fallopian tube involvement. XGI was also seen associated with other primary lesions of FGT like high-grade serous carcinoma(7.5 %) and mature cystic teratoma (7.5 %), while non-neoplastic associations included tubal gestation, foreign body, and Aspergillus infection in the ovary. Histologically, the infiltrate comprised of chronic inflammatory cells in all cases with additional acute inflammatory cells(60 %). Multinucleated giant cells were seen in 40 % of cases. Urine culture showed bacterial colonies in 23 % of cases. CONCLUSIONS: XGI of FGT is an extremely rare lesion and can present as isolated lesions or in association with other primary lesions of FGT. Adnexal involvement was more common than uterine XGI. It is essential to recognize this lesion because it can mimic malignancy and has a destructive nature.

Epidermodysplasia Verruciformis: A Study of Clinicopathologic Features, Biomarkers, and Associated Malignancies in Indian Patients.

BACKGROUND: Epidermodysplasia verruciformis (EDV) is a rare cutaneous manifestation of human papilloma virus infection, which has a potential for malignant transformation. The characteristic histologic features of EDV may not always be present and may often be overlooked. The use of a panel of novel biomarkers may aid in differentiating EDV from their clinical and pathologic mimics. MATERIAL AND METHODS: We reviewed 20 cases histologically diagnosed as EDV from 2013 to 2022. Sections were reviewed for histopathologic features, and immunohistochemistry for p16 and Ki67 was performed. RESULTS: There were 20 cases, ranging in age from 6 to 52 years with a male predominance. Four patients were immunosuppressed, and 4 patients had a positive family history. The most common presentation was hypopigmented papules and macules. In all the cases, epidermal keratinocytes showed dysmaturation, enlargement, and a blue-gray cytoplasm. These changes were very focal and superficial in 15 cases (75%). Associated malignancies included carcinoma in situ (1), trichilemmoma (2), and trichilemmal carcinoma (1). The trichilemmal tumors were seen in 2 siblings. p16 was expressed in the parabasal and basal layers in 7 of 17 cases (41%), in keratinocytes with and without inclusions. Ki67 was increased and localized to suprabasal and parabasal keratinocytes in 15 of 17 cases (88%). CONCLUSION: Although striking and characteristic, the keratinocyte changes are often focal and superficial, requiring multiple step-sections. Association of EDV with familial trichilemmal neoplasms is a novel finding requiring further genetic testing. In cases of clinically suspected EDV with negative histopathologic findings, p16 and Ki67 seem useful as adjunct biomarkers and could serve as cost-effective alternatives to genotyping.

Drugging the efferocytosis process: concepts and opportunities.

The daily removal of billions of apoptotic cells in the human body via the process of efferocytosis is essential for homeostasis. To allow for this continuous efferocytosis, rapid phenotypic changes occur in the phagocytes enabling them to engulf and digest the apoptotic cargo. In addition, efferocytosis is actively anti-inflammatory and promotes resolution. Owing to its ubiquitous nature and the sheer volume of cell turnover, efferocytosis is a point of vulnerability. Aberrations in efferocytosis are associated with numerous inflammatory pathologies, including atherosclerosis, cancer and infections. The recent exciting discoveries defining the molecular machinery involved in efferocytosis have opened many avenues for therapeutic intervention, with several agents now in clinical trials.

Targeting SLC7A11 improves efferocytosis by dendritic cells and wound healing in diabetes.

Chronic non-healing wounds are a major complication of diabetes, which affects 1 in 10 people worldwide. Dying cells in the wound perpetuate the inflammation and contribute to dysregulated tissue repair1-3. Here we reveal that the membrane transporter SLC7A11 acts as a molecular brake on efferocytosis, the process by which dying cells are removed, and that inhibiting SLC7A11 function can accelerate wound healing. Transcriptomics of efferocytic dendritic cells in mouse identified upregulation of several SLC7 gene family members. In further analyses, pharmacological inhibition of SLC7A11, or deletion or knockdown of Slc7a11 using small interfering RNA enhanced efferocytosis in dendritic cells. Slc7a11 was highly expressed in dendritic cells in skin, and single-cell RNA sequencing of inflamed skin showed that Slc7a11 was upregulated in innate immune cells. In a mouse model of excisional skin wounding, inhibition or loss of SLC7A11 expression accelerated healing dynamics and reduced the apoptotic cell load in the wound. Mechanistic studies revealed a link between SLC7A11, glucose homeostasis and diabetes. SLC7A11-deficient dendritic cells were dependent on aerobic glycolysis using glucose derived from glycogen stores for increased efferocytosis; also, transcriptomics of efferocytic SLC7A11-deficient dendritic cells identified increased expression of genes linked to gluconeogenesis and diabetes. Further, Slc7a11 expression was higher in the wounds of diabetes-prone db/db mice, and targeting SLC7A11 accelerated their wound healing. The faster healing was also linked to the release of the TGFß family member GDF15 from efferocytic dendritic cells. In sum, SLC7A11 is a negative regulator of efferocytosis, and removing this brake improves wound healing, with important implications for wound management in diabetes.

A20 deficiency in myeloid cells protects mice from diet-induced obesity and insulin resistance due to increased fatty acid metabolism.

Obesity-induced inflammation is a major driving force in the development of insulin resistance, type 2 diabetes (T2D), and related metabolic disorders. During obesity, macrophages accumulate in the visceral adipose tissue, creating a low-grade inflammatory environment. Nuclear factor κB (NF-κB) signaling is a central coordinator of inflammatory responses and is tightly regulated by the anti-inflammatory protein A20. Here, we find that myeloid-specific A20-deficient mice are protected from diet-induced obesity and insulin resistance despite an inflammatory environment in their metabolic tissues. Macrophages lacking A20 show impaired mitochondrial respiratory function and metabolize more palmitate both in vitro and in vivo. We hypothesize that A20-deficient macrophages rely more on palmitate oxidation and metabolize the fat present in the diet, resulting in a lean phenotype and protection from metabolic disease. These findings reveal a role for A20 in regulating macrophage immunometabolism.

Microbes exploit death-induced nutrient release by gut epithelial cells.

Regulated cell death is an integral part of life, and has broad effects on organism development and homeostasis1. Malfunctions within the regulated cell death process, including the clearance of dying cells, can manifest in diverse pathologies throughout various tissues including the gastrointestinal tract2. A long appreciated, yet elusively defined relationship exists between cell death and gastrointestinal pathologies with an underlying microbial component3-6, but the direct effect of dying mammalian cells on bacterial growth is unclear. Here we advance a concept that several Enterobacteriaceae, including patient-derived clinical isolates, have an efficient growth strategy to exploit soluble factors that are released from dying gut epithelial cells. Mammalian nutrients released after caspase-3/7-dependent apoptosis boosts the growth of multiple Enterobacteriaceae and is observed using primary mouse colonic tissue, mouse and human cell lines, several apoptotic triggers, and in conventional as well as germ-free mice in vivo. The mammalian cell death nutrients induce a core transcriptional response in pathogenic Salmonella, and we identify the pyruvate formate-lyase-encoding pflB gene as a key driver of bacterial colonization in three contexts: a foodborne infection model, a TNF- and A20-dependent cell death model, and a chemotherapy-induced mucositis model. These findings introduce a new layer to the complex host-pathogen interaction, in which death-induced nutrient release acts as a source of fuel for intestinal bacteria, with implications for gut inflammation and cytotoxic chemotherapy treatment.

Metabolites released from apoptotic cells act as tissue messengers.

Caspase-dependent apoptosis accounts for approximately 90% of homeostatic cell turnover in the body1, and regulates inflammation, cell proliferation, and tissue regeneration2-4. How apoptotic cells mediate such diverse effects is not fully understood. Here we profiled the apoptotic metabolite secretome and determined its effects on the tissue neighbourhood. We show that apoptotic lymphocytes and macrophages release specific metabolites, while retaining their membrane integrity. A subset of these metabolites is also shared across different primary cells and cell lines after the induction of apoptosis by different stimuli. Mechanistically, the apoptotic metabolite secretome is not simply due to passive emptying of cellular contents and instead is a regulated process. Caspase-mediated opening of pannexin 1 channels at the plasma membrane facilitated the release of a select subset of metabolites. In addition, certain metabolic pathways continued to remain active during apoptosis, with the release of only select metabolites from a given pathway. Functionally, the apoptotic metabolite secretome induced specific gene programs in healthy neighbouring cells, including suppression of inflammation, cell proliferation, and wound healing. Furthermore, a cocktail of apoptotic metabolites reduced disease severity in mouse models of inflammatory arthritis and lung-graft rejection. These data advance the concept that apoptotic cells are not inert cells waiting for removal, but instead release metabolites as 'good-bye' signals to actively modulate outcomes in tissues.

Nucleolar fibrillarin is an evolutionarily conserved regulator of bacterial pathogen resistance.

Innate immunity is the first line of defense against infections. Pathways regulating innate responses can also modulate other processes, including stress resistance and longevity. Increasing evidence suggests a role for the nucleolus in regulating cellular processes implicated in health and disease. Here we show the highly conserved nucleolar protein, fibrillarin, is a vital factor regulating pathogen resistance. Fibrillarin knockdown enhances resistance in C. elegans against bacterial pathogens, higher levels of fibrillarin induce susceptibility to infection. Pathogenic infection reduces nucleolar size, ribsosomal RNA, and fibrillarin levels. Genetic epistasis reveals fibrillarin functions independently of the major innate immunity mediators, suggesting novel mechanisms of pathogen resistance. Bacterial infection also reduces nucleolar size and fibrillarin levels in mammalian cells. Fibrillarin knockdown prior to infection increases intracellular bacterial clearance, reduces inflammation, and enhances cell survival. Collectively, these findings reveal an evolutionarily conserved role of fibrillarin in infection resistance and suggest the nucleolus as a focal point in innate immune responses.

Calcium dynamics in cardiac excitatory and non-excitatory cells and the role of gap junction.

Calcium ions aid in the generation of action potential in myocytes and are responsible for the excitation-contraction coupling of heart. The heart muscle has specialized patches of cells, called excitatory cells (EC) such as the Sino-atrial node cells capable of auto-generation of action potential and cells which receive signals from the excitatory cells, called non-excitatory cells (NEC) such as cells of the ventricular and auricular walls. In order to understand cardiac calcium homeostasis, it is, therefore, important to study the calcium dynamics taking into account both types of cardiac cells. Here we have developed a model to capture the calcium dynamics in excitatory and non-excitatory cells taking into consideration the gap junction mediated calcium ion transfer from excitatory cell to non-excitatory cell. Our study revealed that the gap junctional coupling between excitatory and non-excitatory cells plays important role in the calcium dynamics. It is observed that any reduction in the functioning of gap junction may result in abnormal calcium oscillations in NEC, even when the calcium dynamics is normal in EC cell. Sensitivity of gap junction is observed to be independent of the pacing rate and hence a careful monitoring is required to maintain normal cardiomyocyte condition. It also highlights that sarcoplasmic reticulum may not be always able to control the amount of cytoplasmic calcium under the condition of calcium overload.

A mathematical model predicting host mitochondrial pyruvate transporter activity to be a critical regulator of Mycobacterium tuberculosis pathogenicity.

Modulation of host metabolic machinery by Mycobacterium tuberculosis is a well established phenomenon. In our earlier study (Mehrotra et al., 2014), we observed a marked increase in acetyl-CoA levels in cells bearing virulent M. tuberculosis infections compared to host cells harbouring avirulent infections. The difference was observed inspite of similar levels of total host cellular pyruvate in both infection types. The present study aimed in capturing the cause for such a phenomenon that defines the pathogenicity of M. tuberculosis. Through mathematical model, we dissected the relative importance of virulence mediated effect on Pyruvate dehydrogenase (PDH) activity, rate of acetyl-CoA consumption and mitochondrial pyruvate transporter (MPC) activity in causing the observed outcomes. Simulation results exhibit MPC to be the key regulatory junction perturbed by virulent strains of M. tuberculosis leading to alteration of mitochondrial metabolic flux and regulation of acetyl-CoA formation. As an experimental validation, drug mediated inhibition of MPC activity was sufficient to reduce virulent bacillary loads, pointing towards a possible mechanistic target for drug discovery.

Mycobacterial escape from macrophage phagosomes to the cytoplasm represents an alternate adaptation mechanism.

Survival of Mycobacterium tuberculosis (Mtb) within the host macrophage is mediated through pathogen-dependent inhibition of phagosome-lysosome fusion, which enables bacteria to persist within the immature phagosomal compartment. By employing ultrastructural examination of different field isolates supported by biochemical analysis, we found that some of the Mtb strains were in fact poorly adapted for subsistence within endocytic vesicles of infected macrophages. Instead, through a mechanism involving activation of host cytosolic phospholipase A2, these bacteria rapidly escaped from phagosomes, and established residence in the cytoplasm of the host cell. Interestingly, by facilitating an enhanced suppression of host cellular autophagy, this translocation served as an alternate virulence acquisition mechanism. Thus, our studies reveal plasticity in the adaptation strategies employed by Mtb, for survival in the host macrophage.

Pathogenicity of Mycobacterium tuberculosis is expressed by regulating metabolic thresholds of the host macrophage.

The success of Mycobacterium tuberculosis as a pathogen derives from its facile adaptation to the intracellular milieu of human macrophages. To explore this process, we asked whether adaptation also required interference with the metabolic machinery of the host cell. Temporal profiling of the metabolic flux, in cells infected with differently virulent mycobacterial strains, confirmed that this was indeed the case. Subsequent analysis identified the core subset of host reactions that were targeted. It also elucidated that the goal of regulation was to integrate pathways facilitating macrophage survival, with those promoting mycobacterial sustenance. Intriguingly, this synthesis then provided an axis where both host- and pathogen-derived factors converged to define determinants of pathogenicity. Consequently, whereas the requirement for macrophage survival sensitized TB susceptibility to the glycemic status of the individual, mediation by pathogen ensured that the virulence properties of the infecting strain also contributed towards the resulting pathology.