Computational Modeling of T Cell Hypersensitivity during Coronavirus Infections Leading to Autoimmunity and Lethality.

The human angiotensin-converting enzyme 2 (hACE2) receptor is the primary receptor for SARS-CoV-2 infection. However, the presence of alternative receptors such as the transmembrane glycoprotein CD147 has been proposed as a potential route for SARS-CoV-2 infection. The outcomes of SARS-CoV-2 spike protein binding to receptors have been shown to vary among individuals. Additionally, some patients infected with SARS-CoV-2 develop autoimmune responses. Given that CD147 is involved in the hyperactivation of memory T cells resulting in autoimmunity, we investigated the interaction of the SARS-CoV-2 viral spike protein with CD147 receptor and retinal specific CD147 Ig0 domain in silico using molecular docking and molecular dynamics (MD) simulations. The results indicated that binding involves two critical residues Lys63 and Asp65 in a ubiquitous CD147 isoform, potentially leading to the hyperactivation of T cells for only SARS-CoV-2, but not for SARS-CoV or MERS-CoV. Overall binding was confirmed by docking simulations. Next, MD analyses were completed to verify the docking poses. Polar interactions suggested that the interaction via Lys63 and Asp65 might be one of the determinants associated with severe COVID-19 outcomes. Neither did SARS-CoV nor MERS-CoV bind to these two critical residues when molecular docking analyses were performed. Interestingly, SARS-CoV was able to bind to CD147 with a lower affinity (-4.5 kcal/mol) than SARS-CoV-2 (-5.6 kcal/mol). Furthermore, Delta and Omicron variants of SARS-CoV-2 did not affect the polar interactions with Lys63 and Asp65 in CD147. This study further strengthens the link between SARS-CoV-2 infection and autoimmune responses and provides novel insights for prudent antiviral drug designs for COVID-19 treatment that have implications in the prevention of T cell hyperactivation.

Correction: Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock.

[This corrects the article DOI: 10.1371/journal.pone.0152385.].

Interferon Regulatory Factor 6 Has a Protective Role in the Host Response to Endotoxic Shock.

Interferon Regulatory Factor (IRF) 6, a member of the IRF family, is essential for epidermal and orofacial embryonic development. Irf6 is strongly expressed in keratinocytes, in which it regulates epidermal proliferation, differentiation, and migration. A recent role for Irf6 in Toll-like receptor 2-dependent chemokine gene expression was also reported in an epithelial cell line. However, a function for Irf6 in innate immune cells was not previously reported. In the present study, we investigated the expression and function of Irf6 in bone marrow-derived neutrophils and macrophages. We show here, using a conditional knockout of Irf6 in lysosymeM expressing cells, that Irf6 is required for resistance to LPS-induced endotoxic shock. In addition, Irf6-deficient bone marrow-derived neutrophils exhibited increased chemotactic index and velocity compared with wild-type cells in vitro. TLR4-specific KC and IL6 secretions were upregulated in Irf6-deficient bone marrow-derived macrophages in vitro. These cells also exhibited an increased level of phosphorylated IkBa. Collectively, our findings suggest a role for Irf6 in the resistance to endotoxic shock due to NFk-B-mediated alteration of cytokine production.

Cutting edge: Nlrp10 is essential for protective antifungal adaptive immunity against Candida albicans.

Nucleotide-binding domain leucine-rich repeat containing receptors (NLRs) are cytosolic receptors that initiate immune responses to sterile and infectious insults to the host. Studies demonstrated that Nlrp3 is critical for the control of Candida albicans infections and in the generation of antifungal Th17 responses. In this article, we show that the NLR family member Nlrp10 also plays a unique role in the control of disseminated C. albicans infection in vivo. Nlrp10-deficient mice had increased susceptibility to disseminated candidiasis, as indicated by decreased survival and increased fungal burdens. In contrast to Nlrp3, Nlrp10 deficiency did not affect innate proinflammatory cytokine production from macrophages and dendritic cells challenged with C. albicans. However, Nlrp10-deficient mice displayed a profound defect in Candida-specific Th1 and Th17 responses. These results demonstrate a novel role for Nlrp10 in the generation of adaptive immune responses to fungal infection.

NLRP10 is a NOD-like receptor essential to initiate adaptive immunity by dendritic cells.

NLRs (nucleotide-binding domain leucine-rich-repeat-containing receptors; NOD-like receptors) are a class of pattern recognition receptor (PRR) that respond to host perturbation from either infectious agents or cellular stress. The function of most NLR family members has not been characterized and their role in instructing adaptive immune responses remains unclear. NLRP10 (also known as PYNOD, NALP10, PAN5 and NOD8) is the only NLR lacking the putative ligand-binding leucine-rich-repeat domain, and has been postulated to be a negative regulator of other NLR members, including NLRP3 (refs 4-6). We did not find evidence that NLRP10 functions through an inflammasome to regulate caspase-1 activity nor that it regulates other inflammasomes. Instead, Nlrp10(-/-) mice had a profound defect in helper T-cell-driven immune responses to a diverse array of adjuvants, including lipopolysaccharide, aluminium hydroxide and complete Freund's adjuvant. Adaptive immunity was impaired in the absence of NLRP10 because of a dendritic cell (DC) intrinsic defect in emigration from inflamed tissues, whereas upregulation of DC costimulatory molecules and chemotaxis to CCR7-dependent and -independent ligands remained intact. The loss of antigen transport to the draining lymph nodes by a subset of migratory DCs resulted in an almost absolute loss in naive CD4(+) T-cell priming, highlighting the critical link between diverse innate immune stimulation, NLRP10 activity and the immune function of mature DCs.

Quantitation of SPLUNC1 in saliva with an xMAP particle-based antibody capture and detection immunoassay.

The short palate lung and nasal epithelial clone 1 (SPLUNC1) protein may be differentially expressed in oral infections, oral inflammatory disorders, or oral malignancies and may be involved in innate immune responses in the oral cavity. However, the actual concentration of SPLUNC1 in saliva has not previously been determined. In this study, we determined the concentrations of SPLUNC1 in saliva using a particle-based antibody capture and detection immunoassay. A commercial goat anti-rhSPLUNC1 polyclonal antibody (AF1897) was linked to fluorescent polystyrene microspheres and used as the capture antibody. A commercial mouse IgG2b anti-rhSPLUNC1 monoclonal antibody (MAB1897) was biotinylated and used as the detection antibody. Western blot and 2-dimensional fluorescence difference gel electrophoresis (2-D DIGE) analysis of immunoprecipitated rhSPLUNC1 and SPLUNC1 from saliva were used to show that the capture AF1897 and detection MAB1897 antibodies both recognized SPLUNC1. Protein concentrations in saliva from 20 subjects ranged from 0.9 to 23.9mg/ml; SPLUNC1 concentrations ranged from 34.7ng/ml to 13.8µg/ml; and SPLUNC concentrations normalized per mg of total salivary protein ranged from 4.7ng/ml to 5.3µg/ml. These results show that SPLUNC1 is detected in saliva in a variety of concentrations. This immunoassay may prove to be useful in determining the concentration of SPLUNC1 in saliva for assessing its role in the pathogenesis of oral infections, oral inflammatory disorders, or oral malignancies.

Influence of smoking on gingival crevicular fluid cytokines in severe chronic periodontitis.

AIM: The aim of this study was to compare the expression of 22 chemokines and cytokines in gingival crevicular fluid (GCF) from smokers and non-smokers with periodontitis and periodontally healthy control subjects. MATERIALS AND METHODS: Forty subjects with generalized severe chronic periodontitis (20 smokers and 20 non-smokers) and 12 periodontally healthy control subjects participated in this study. Four diseased and two healthy sites were selected from each of the periodontitis subjects. GCF samples were collected and cytokines analysed utilizing a multiplexed immunoassay (Luminex(®) ). Statistical analyses employed non-parametric tests including the Mann-Whitney and Wilcoxon matched-pairs signed-rank tests. RESULTS: Compared with healthy control subjects, GCF in subjects with chronic periodontitis contained significantly higher amounts of interleukin (IL)-1α, IL-1ß, IL-6, IL-12(p40) (pro-inflammatory cytokines); IL-8, macrophage chemotactic protein (MCP)-1, macrophage inflammatory protein (MIP)-1α, regulated on activation normal T-cell expressed and secreted (RANTES) (chemokines); IL-2, IFN-γ, IL-3, IL-4 (Th1/Th2 cytokines); IL-15 [regulator of T-cells and natural killer (NK) cells]. Smokers displayed decreased amounts of pro-inflammatory cytokines [IL-1α, IL-6, IL-12(p40)], chemokines (IL-8, MCP-1, MIP-1, RANTES), and regulators of T-cells and NK cells (IL-7, IL-15). CONCLUSIONS: Periodontitis subjects had significantly elevated cytokine and chemokine profiles. Smokers exhibited a decrease in several pro-inflammatory cytokines and chemokines and certain regulators of T-cells and NK-cells. This reflects the immunosuppressant effects of smoking which may contribute to an enhanced susceptibility to periodontitis.

Fungal pathogen recognition by the NLRP3 inflammasome.

The relationship between host and opportunistic pathogen is a tenuous one and an injudicious response to pathogen-associated molecular patterns may result in a hostile environment to potentially beneficial commensal organisms. Therefore, discrimination between pathogenic forms, causing cellular damage, and innocuous commensal forms of microbes is critical in maintaining homeostasis. The NLRP3 inflammasome has recently been identified as playing an important role in recognition of the fungal pathogen Candida albicans. Here we will review these findings and discuss the role of the NLRP3 inflammasome in initiating an innate immune response to invasive C. albicans.

Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome.

The proinflammatory cytokine IL-1beta plays an important role in antifungal immunity; however, the mechanisms by which fungal pathogens trigger IL-1beta secretion are unclear. In this study we show that infection with Candida albicans is sensed by the Nlrp3 inflammasome, resulting in the subsequent release of IL-1beta. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome, as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL- 1beta secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans, which is consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.

The NLRP3 inflammasome: a sensor of immune danger signals.

The innate immune system senses danger signals via evolutionary conserved receptors. The nucleotide-binding domain leucine-rich repeat containing receptor (NLR) family is a group of intracellular receptors that drive a wide variety of inflammatory responses. A number of the NLR family members can form inflammasomes, which are multiprotein complexes that can activate caspase-1 and ultimately lead to the processing and secretion of interleukin (IL)-1beta, IL-18 and IL-33. One of the best-studied members of the NLR family is NLRP3 for which a number of divergent activators have recently been described. These and other studies examining the NLRP3 inflammasome will be discussed in this review.

Human beta-defensin 3 binds to hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis, and attenuates a pro-inflammatory cytokine response.

Regulatory mechanisms in mucosal secretions and tissues recognize antigens and attenuate pro-inflammatory cytokine responses. Here, we asked whether human beta-defensin 3 (HBD3) serves as an upstream suppressor of cytokine signaling that binds and attenuates pro-inflammatory cytokine responses to recombinant hemagglutinin B (rHagB), a non-fimbrial adhesin from Porphyromonas gingivalis strain 381. We found that HBD3 binds to immobilized rHagB and produces a significantly higher resonance unit signal in surface plasmon resonance spectroscopic analysis, than HBD2 and HBD1 that are used as control defensins. Furthermore, we found that HBD3 significantly attenuates (P<0.05) the interleukin (IL)-6, IL-10, granulocyte macrophage colony stimulating factor (GM-CSF) and tumor-necrosis factor-alpha (TNF-alpha) responses induced by rHagB in human myeloid dendritic cell culture supernatants and the extracellular signal-regulated kinases (ERK 1/2) response in human myeloid dendritic cell lysates. Thus, HBD3 binds rHagB and this interaction may be an important initial step to attenuate a pro-inflammatory cytokine response and an ERK 1/2 response.

Correlation between beta-defensin expression and induction profiles in gingival keratinocytes.

Human beta-defensins are antimicrobial peptides produced by epithelial cells. To date, 28 beta-defensins have been described and the expression of a select few has been classified as constitutive or inducible. Most studies have evaluated expression and regulation using a limited number of primary cell cultures or immortalized cell lines. The goal of this study was to quantitatively assess the in vitro expression and inducibility profiles of human beta-defensins, HBD-1, HBD-2, and HBD-3 across a number of primary gingival keratinocyte cultures. Cultured cells from 14 human subjects were stimulated with interleukin-1 beta (IL-1beta), IL-2, IL-6, IL-8, IL-12, tumor necrosis factor alpha (TNF-alpha), gamma interferon (IFN-gamma) or Escherichia coli lipopolysaccharide (LPS) and analyzed by reverse transcription (RT)-PCR. A subset of cultures were quantitatively assessed by real-time PCR. HBD-1 presented the highest and most heterogeneous expression at the basal level (non-stimulated) as compared to expression of HBD-2 and HBD-3, which was significantly lower and homogeneous. IFN-gamma was a primary inducer for HBD-1 and HBD-3, while IL-1beta and TNF-alpha were primary inducers for HBD-2. Sporadic induction was seen for IL-2, IL-6 and LPS. Synergistic expression was seen when various cytokines were combined. Interestingly, the induction potential of each beta-defensin was directly correlated to its basal expression. An inhibitor of JAK2 kinase (Janus kinase), down-regulated IFN-gamma-induced HBD-1 and HBD-3 expression, suggesting a role for the JAK/signal transducer and activator of transcription (STAT) signaling pathway in their expression. HBD-2 protein expression of supernatants and cell lysates paralleled mRNA expression. The results suggest that beta-defensin expression and induction in gingival keratinocytes is similar to that seen in other tissue. However, the novel finding of considerable variation among induction levels and the correlation of the induction with basal expression suggests that these innate response elements may play a key role in susceptibility or resistance to disease in the oral cavity.

Candida albicans switch phenotypes display differential levels of fitness.

BACKGROUND: Candidiasis is a significant cause of morbidity and mortality in immunosuppressed individuals. Currently, there are limited treatment options for this opportunistic infection and there have been many reports in the literature of increased resistance to treatment. There is evidence to suggest that phenotypic switching of Candida albicans may play a role in this resistance. It was therefore, the purpose of this study to assess the variability among C. albicans switch phenotypes isolated from a single strain. MATERIAL/METHODS: Four switch phenotypes from a single strain of Candida albicans isolated from an HIV+ individual were evaluated for growth rates, post-antifungal effects in the presence of fluconazole, fluconazole uptake and morphological changes after fluconazole exposure. RESULTS: We found that, overall, smooth white and very wrinkled grew significantly faster then either ring or heavily myceliated and were most drastically affected by 4X and 6X MIC concentrations of fluconazole over a 24 hour period. When all four phenotypes were exposed to 64. CONCLUSIONS: These results suggest that switch phenotypes from Candida albicans serve different roles in providing adaptability and survivability under differing conditions.

Expression of beta-defensins in gingival health and in periodontal disease.

BACKGROUND: Human beta-defensins (HBDs) are epithelial-derived antimicrobial peptides. The expression of three HBDs (HBD-1, HBD-2, and HBD-3) has been reported in oral mucosa, gingiva, and salivary glands. However, their role in protection against oral infections is not well understood. This study examined the expression of HBD-1, HBD-2, and HBD-3 mRNAs in gingival health and periodontal disease. METHODS: Gingival tissue discarded from periodontal procedures was obtained from 20 periodontally healthy and 29 periodontally diseased sites. Total RNA was isolated, and expression of beta-defensins was analyzed by both 35-cycle and semiquantitative (25-cycle) reverse transcription-polymerase chain reaction (RT-PCR). The level of expression was assigned ordinal scores of no expression, low expression, or high expression. mRNA expression was compared between healthy and diseased groups using exact tests of homogeneity and Cochran-Mantel-Haenszel tests; associations among the variables were assessed using exact tests and Kendall's tau-b statistic. RESULTS: All 49 samples demonstrated basal mRNA expression of HBD-1, HBD-2, and HBD-3. Significantly higher levels of HBD-3 (P = 0.012) expression were found in the healthy tissues as compared to the diseased ones. There was also a suggestion of higher expression of HBD-2 in the healthy tissues (P = 0.12). Levels of HBD-1, HBD-2, and HBD-3 mRNA expression were correlated with one another (P < 0.001). CONCLUSIONS: High levels of HBD-3 mRNA expression in healthy tissues suggest a potentially important protective role for defensins in the host immune response to infection by periodontal pathogens.

Human beta-defensins 2 and 3 demonstrate strain-selective activity against oral microorganisms.

Human beta-defensins 2 and 3 (HBD-2 and HBD-3) are inducible peptides present at sites of infection in the oral cavity. A few studies have reported broad-spectrum antimicrobial activity for both peptides. However, no comprehensive study has thoroughly investigated their potential against oral pathogens. The purpose of this study was to test the effectiveness of HBD-2 and HBD-3 against a collection of oral organisms (Actinobacillus actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis, Peptostreptococcus micros, Actinomyces naeslundii, Actinomyces israelii, Streptococcus sanguis, Streptococcus mutans, Candida tropicalis, Candida parapsilosis, Candida krusei, Candida glabrata, and Candida albicans). Radial diffusion assays were used to test HBD-2 and HBD-3 activities against at least three strains of each species. There was significant variability in MICs, which was strain specific rather than species specific. MICs ranged from 3.9 to >250 micro g/ml for HBD-2 and from 1.4 to >250 micro g/ml for HBD-3. HBD-3 demonstrated greater antimicrobial activity and was effective against a broader array of organisms. Overall, aerobes were 100% susceptible to HBD-2 and HBD-3, whereas only 21.4 and 50% of the anaerobes were susceptible to HBD-2 and HBD-3, respectively. HBD-2 and HBD-3 also demonstrated strain-specific activity against the Candida species evaluated. Interestingly, an association between HBD-2 and HBD-3 activities was noted. This suggests that the two peptides may have similar mechanisms yet utilize distinct pathways. The lack of activity against specific anaerobic strains and Candida warrants further investigation of the potential resistance mechanisms of these organisms. Finally, the significant variability between strains underlies the importance of testing multiple strains when evaluating activities of antimicrobial peptides.

Racial distribution of Candida dubliniensis colonization among South Africans.

Candida dubliniensis is a yeast species that has only recently been differentiated from Candida albicans. C. dubliniensis colonization was initially associated with human immunodeficiency virus (HIV)-positive individuals. Because of the large proportion of AIDS patients in South Africa, we tested the generality of this association by assessing the prevalence of C. dubliniensis colonization among 253 black HIV-positive individuals, 66 healthy black individuals, 22 white HIV-positive individuals, and 55 healthy white individuals in South Africa carrying germ tube-positive yeasts in their oral cavities. Molecular fingerprinting with Ca3, a complex DNA fingerprinting probe specific for C. albicans, and Cd25, a complex DNA fingerprinting probe specific for C. dubliniensis, provides the first conclusive evidence of the existence of C. dubliniensis among South African clinical yeast isolates and reveals a higher relative prevalence of this species among white healthy individuals (16%) than among HIV-positive white individuals (9%), black healthy individuals (0%), and black HIV-positive individuals (1.5%). A cluster analysis separated South African C. dubliniensis isolates into two previously described groups, groups I and II, with the majority of isolates clustering in group I. Isolates from white healthy individuals exhibited a higher level of relatedness. A comparison of the C. dubliniensis isolates from South Africa with a general collection of C. dubliniensis isolates collected worldwide revealed no South Africa-specific clade, as has been demonstrated for C. albicans. These results suggest that in South Africa, C. dubliniensis carriage is influenced more by race than by HIV infection status.

Phenotypic switching and filamentation in Candida glabrata.

Candida glabrata switches spontaneously, reversibly and at high frequency among the following four phenotypes distinguishable by graded colony colouration on CuSO(4)-containing agar: white (Wh), light brown (LB), dark brown (DB) and very dark brown (vDB). These phenotypes also differ in a graded fashion in the level of expression of the metallothionein gene MTII (WhvDB), the frequency of switching (Wh>LB>DB>vDB) and colouration on phloxine B-containing agar (Wh>LB>DB>vDB). Switching among the four graded phenotypes is referred to as 'the core switching system'. An additional switch phenotype, 'irregular wrinkle' (IWr), has been identified, which exhibits a highly wrinkled colony morphology. The characteristics of IWr suggest that switching to and from this phenotype represents a second high-frequency switching system. A microscopic analysis revealed that during the first 3 days of colony development, cells in the centres of Wh, LB, DB and vDB colonies expressed almost exclusively the budding yeast phenotype. After 3 days, however, pseudohyphae and cells extending tubes accumulated, so that by 7 days the proportions of these two cellular phenotypes reached 40-50% and 10-20%, respectively. In contrast, IWr colonies were composed almost exclusively of pseudohyphae through the first 6 days of colony development. After 6 days, IWr colonies began to accumulate both budding yeast cells and tubes. The tubes formed by C. glabrata reached lengths of up to six cell diameters, but the tubes did not represent traditional compartmentalized hyphae. Tube growth ended when the tube tip expanded to form a bud. Tubes then functioned as corridors for daughter nucleus migration to the apical bud, and were ultimately left uncompartmentalized and nucleus free. Core switching, pseudohypha formation and tube formation occurred in a majority of 62 tested clinical isolates, demonstrating that these developmental programmes are general characteristics of most strains of C. glabrata.

Ca3 fingerprinting of Candida albicans isolates from human immunodeficiency virus-positive and healthy individuals reveals a new clade in South Africa.

To examine the question of strain specificity in oropharyngeal candidiasis associated with human immunodeficiency virus (HIV) infection, oral samples were collected from 1,196 HIV-positive black South Africans visiting three clinics and 249 Candida albicans isolates were selected for DNA fingerprinting with the complex DNA fingerprinting probe Ca3. A total of 66 C. albicans isolates from healthy black South Africans and 46 from healthy white South Africans were also DNA fingerprinted as controls. Using DENDRON software, a cluster analysis was performed and the identified groups were compared to a test set of isolates from the United States in which three genetic groups (I, II, and III) were previously identified by a variety of genetic fingerprinting methods. All of the characterized South African collections (three from HIV-positive black persons, two from healthy black persons, and one from healthy white persons) included group I, II, and III isolates. In addition, all South African collections included a fourth group (group SA) completely absent in the U.S. collection. The proportion of group SA isolates in HIV-positive and healthy black South Africans was 53% in both cases. The proportion in healthy white South Africans was 33%. In a comparison of HIV-positive patients with and without oropharyngeal symptoms of infection, the same proportions of group I, II, III, and SA isolates were obtained, indicating no shift to a particular group on infection. However, by virtue of its predominance as a commensal and in infections, group SA must be considered the most successful in South Africa. Why group SA isolates represent 53 and 33% of colonizing strains in black and white South Africans and are absent in the U.S. collection represents an interesting epidemiological question.

Carriage frequency, intensity of carriage, and strains of oral yeast species vary in the progression to oral candidiasis in human immunodeficiency virus-positive individuals.

Candida samples were taken over a period of 2 years from 54 human immunodeficiency virus (HIV)-positive asymptomatic subjects to evaluate changes in yeast carriage, intensity of carriage, and genotype over time. Overall, we found that HIV-positive patients with CD4(+)-cell counts of between 200 and 400/microl had significantly more yeast colonization than healthy control subjects. Of the 54 patients, 11 developed thrush. We found that intensity of carriage in these 11 patients increased significantly in the progression from asymptomatic yeast carrier to an episode of oral thrush. Also, the most common yeast species isolated was Candida albicans; however, we did see a number of patients harboring multiple species at the same time. Using the C. albicans-specific probe Ca3, we found that 54% (n = 6) of the 11 patients who developed thrush maintained genetically similar strains throughout the study period, with minor genetic variations in all patients except one. Forty-six percent of these patients had either multiple strains throughout the study period (n = 2), strain replacement (n = 1), or species replacement (n = 2). Of the patients who had multiple strains, one (I4) was infected by two different strains of Candida dubliniensis distinguished by a recently developed species-specific probe. These results suggest that commensal strains colonizing HIV-positive individuals can undergo alterations prior to producing an episode of thrush.