Evaluation of SARS-CoV-2 interferon gamma release assay in BNT162b2 vaccinated healthcare workers.

To predict protective immunity to SARS-CoV-2, cellular immunity seems to be more sensitive than humoral immunity. Through an Interferon-Gamma (IFN-γ) Release Assay (IGRA), we show that, despite a marked decrease in total antibodies, 94.3% of 123 healthcare workers have a positive cellular response 6 months after inoculation with the 2nd dose of BNT162b2 vaccine. Despite the qualitative relationship found, we did not observe a quantitative correlation between IFN-γ and IgG levels against SARS-CoV-2. Using stimulated whole blood from a subset of participants, we confirmed the specific T-cell response to SARS-CoV-2 by dosing elevated levels of the IL-6, IL-10 and TNF-α. Through a 20-month follow-up, we found that none of the infected participants had severe COVID-19 and that the first positive cases were only 12 months after the 2nd dose inoculation. Future studies are needed to understand if IGRA-SARS-CoV-2 can be a powerful diagnostic tool to predict future COVID-19 severe disease, guiding vaccination policies.

Peritoneal B1 and B2 cells respond differently to LPS and IL-21 stimulation.

Peritoneal B cells can be divided into B1 cells (CD11b+CD19+) and B2 cells (CD11b-CD19+) based on CD11b expression. B1 cells play a crucial role in the innate immune response by producing natural antibodies and cytokines. B2 cells share similar traits with B1 cells, influenced by the peritoneal environment. However, the response of both B1 and B2 cells to the same stimuli in the peritoneum remains uncertain. We isolated peritoneal B1 and B2 cells from mice and assessed differences in Interleukin-10(IL-10) secretion, apoptosis, and surface molecule expression following exposure to LPS and Interleukin-21(IL-21). Our findings indicate that B1 cells are potent IL-10 producers, possessing surface molecules with an IgMhiCD43+CD21low profile, and exhibit a propensity for apoptosis in vitro. Conversely, B2 cells exhibit lower IL-10 production and surface markers characterized as IgMlowCD43-CD21hi, indicative of some resistance to apoptosis. LPS stimulates MAPK phosphorylation in B1 and B2 cells, causing IL-10 production. Furthermore, LPS inhibits peritoneal B2 cell apoptosis by enhancing Bcl-xL expression. Conversely, IL-21 has no impact on IL-10 production in these cells. Nevertheless, impeding STAT3 phosphorylation permits IL-21 to increase IL-10 production in peritoneal B cells. Moreover, IL-21 significantly raises apoptosis levels in these cells, a process independent of STAT3 phosphorylation and possibly linked to reduced Bcl-xL expression. This study elucidates the distinct functional and response profiles of B1 and B2 cells in the peritoneum to stimuli like LPS and IL-21, highlighting their differential roles in immunological responses and B cell diversity.

The Role of Coinhibitory Receptors in B Cell Dysregulation in SARS-CoV-2-Infected Individuals with Severe Disease.

Severe SARS-CoV-2 infection is associated with significant immune dysregulation involving different immune cell subsets. In this study, when analyzing critically ill COVID-19 patients versus those with mild disease, we observed a significant reduction in total and memory B cell subsets but an increase in naive B cells. Moreover, B cells from COVID-19 patients displayed impaired effector functions, evidenced by diminished proliferative capacity, reduced cytokine, and Ab production. This functional impairment was accompanied by an increased apoptotic potential upon stimulation in B cells from severely ill COVID-19 patients. Our further studies revealed the expansion of B cells expressing coinhibitory molecules (PD-1, PD-L1, TIM-1, VISTA, CTLA-4, and Gal-9) in intensive care unit (ICU)-admitted patients but not in those with mild disease. The coinhibitory receptor expression was linked to altered IgA and IgG expression and increased the apoptotic capacity of B cells. Also, we found a reduced frequency of CD24hiCD38hi regulatory B cells with impaired IL-10 production. Our mechanistic studies revealed that the upregulation of PD-L1 was linked to elevated plasma IL-6 levels in COVID-19 patients. This implies a connection between the cytokine storm and altered B cell phenotype and function. Finally, our metabolomic analysis showed a significant reduction in tryptophan but elevation of kynurenine in ICU-admitted COVID-19 patients. We found that kynurenine promotes PD-L1 expression in B cells, correlating with increased IL-6R expression and STAT1/STAT3 activation. Our observations provide novel insights into the complex interplay of B cell dysregulation, implicating coinhibitory receptors, IL-6, and kynurenine in impaired B cell effector functions, potentially contributing to the pathogenesis of COVID-19.

Maternal inflammation regulates fetal emergency myelopoiesis.

Neonates are highly susceptible to inflammation and infection. Here, we investigate how late fetal liver (FL) mouse hematopoietic stem and progenitor cells (HSPCs) respond to inflammation, testing the hypothesis that deficits in the engagement of emergency myelopoiesis (EM) pathways limit neutrophil output and contribute to perinatal neutropenia. We show that fetal HSPCs have limited production of myeloid cells at steady state and fail to activate a classical adult-like EM transcriptional program. Moreover, we find that fetal HSPCs can respond to EM-inducing inflammatory stimuli in vitro but are restricted by maternal anti-inflammatory factors, primarily interleukin-10 (IL-10), from activating EM pathways in utero. Accordingly, we demonstrate that the loss of maternal IL-10 restores EM activation in fetal HSPCs but at the cost of fetal demise. These results reveal the evolutionary trade-off inherent in maternal anti-inflammatory responses that maintain pregnancy but render the fetus unresponsive to EM activation signals and susceptible to infection.

A molecular signature for IL-10-producing Th1 cells in protozoan parasitic diseases.

Control of visceral leishmaniasis (VL) depends on proinflammatory Th1 cells that activate infected tissue macrophages to kill resident intracellular parasites. However, proinflammatory cytokines produced by Th1 cells can damage tissues and require tight regulation. Th1 cell IL-10 production is an important cell-autologous mechanism to prevent such damage. However, IL-10-producing Th1 (type 1 regulatory; Tr1) cells can also delay control of parasites and the generation of immunity following drug treatment or vaccination. To identify molecules to target in order to alter the balance between Th1 and Tr1 cells for improved antiparasitic immunity, we compared the molecular and phenotypic profiles of Th1 and Tr1 cells in experimental VL caused by Leishmania donovani infection of C57BL/6J mice. We also identified a shared Tr1 cell protozoan signature by comparing the transcriptional profiles of Tr1 cells from mice with experimental VL and malaria. We identified LAG3 as an important coinhibitory receptor in patients with VL and experimental VL, and we reveal tissue-specific heterogeneity of coinhibitory receptor expression by Tr1 cells. We also discovered a role for the transcription factor Pbx1 in suppressing CD4+ T cell cytokine production. This work provides insights into the development and function of CD4+ T cells during protozoan parasitic infections and identifies key immunoregulatory molecules.

Production of an interleukin-10 blocking antibody by genetically engineered macrophages increases cancer cell death in human gastrointestinal tumor slice cultures.

Although it can promote effector T-cell function, the summative effect of interleukin-10 (IL-10) in the tumor microenvironment (TME) appears to be suppressive; therefore, blocking this critical regulatory cytokine has therapeutic potential to enhance antitumor immune function. As macrophages efficiently localize to the TME, we hypothesized that they could be used as a delivery vehicle for drugs designed to block this pathway. To test our hypothesis, we created and evaluated genetically engineered macrophages (GEMs) that produce an IL-10-blocking antibody (αIL-10). Healthy donor human peripheral blood mononuclear cells were differentiated and transduced with a novel lentivirus (LV) encoding BT-063, a humanized αIL-10 antibody. The efficacy of αIL-10 GEMs was assessed in human gastrointestinal tumor slice culture models developed from resected specimens of pancreatic ductal adenocarcinoma primary tumors and colorectal cancer liver metastases. LV transduction led to sustained production of BT-063 by αIL-10 GEMs for at least 21 days. Transduction did not alter GEM phenotype as evaluated by flow cytometry, but αIL-10 GEMs produced measurable quantities of BT-063 in the TME that was associated with an ~5-fold higher rate of tumor cell apoptosis than control.

Increased Serum Interleukin 10 Levels Are Associated with Increased Disease Activity and Increased Risk of Anti-SS-A/Ro Antibody Positivity in Patients with Systemic Lupus Erythematosus.

Interleukin 10 (IL-10) plays a role in inflammation and cell-type responses. The anti-SS-A/Ro antibody contributes to leucopenia, and cutaneous and neonatal lupus. OBJECTIVES: To evaluate the association between serum IL-10 levels and autoantibodies, disease activity and organ involvement in systemic lupus erythematosus (SLE) patients. PATIENTS AND METHODS: We studied 200 SLE patients and 50 controls. We analyzed organ involvement, disease activity, serum IL-10 and interleukin-6 (IL-6) levels, and antinuclear and antiphospholipid antibody profiles. RESULTS: Serum IL-10 and IL-6 levels were higher in SLE patients than in controls (all p < 0.00001). Serum IL-10 levels were positively correlated with IL-6 (p < 0.00001), CRP (p < 0.00001), fibrinogen (p = 0.003), and ESR (p < 0.00001), and negatively correlated with hemoglobin (p = 0.0004) and lymphocytes (p = 0.01). Serum IL-6 levels were positively correlated with CRP (p < 0.00001), fibrinogen (p = 0.001), and ESR (p < 0.00001); and negatively correlated with hemoglobin (p = 0.008) and lymphocytes (p = 0.03). Elevated serum IL-10 levels were associated with an increased risk of anti-SS-A/Ro antibody positivity (p = 0.03). Elevated serum IL-6 levels were associated with an increased risk of heart (p = 0.007) and lung (p = 0.04) involvement. CONCLUSIONS: In SLE patients, increased serum IL-10 levels were associated with increased disease activity and risk of anti-SS-A/Ro antibody positivity.

Depressive-like Behavior Is Accompanied by Prefrontal Cortical Innate Immune Fatigue and Dendritic Spine Losses after HIV-1 Tat and Morphine Exposure.

Opioid use disorder (OUD) and HIV are comorbid epidemics that can increase depression. HIV and the viral protein Tat can directly induce neuronal injury within reward and emotionality brain circuitry, including the prefrontal cortex (PFC). Such damage involves both excitotoxic mechanisms and more indirect pathways through neuroinflammation, both of which can be worsened by opioid co-exposure. To assess whether excitotoxicity and/or neuroinflammation might drive depressive behaviors in persons infected with HIV (PWH) and those who use opioids, male mice were exposed to HIV-1 Tat for eight weeks, given escalating doses of morphine during the last two weeks, and assessed for depressive-like behavior. Tat expression decreased sucrose consumption and adaptability, whereas morphine administration increased chow consumption and exacerbated Tat-induced decreases in nesting and burrowing-activities associated with well-being. Across all treatment groups, depressive-like behavior correlated with increased proinflammatory cytokines in the PFC. Nevertheless, supporting the theory that innate immune responses adapt to chronic Tat exposure, most proinflammatory cytokines were unaffected by Tat or morphine. Further, Tat increased PFC levels of the anti-inflammatory cytokine IL-10, which were exacerbated by morphine administration. Tat, but not morphine, decreased dendritic spine density on layer V pyramidal neurons in the anterior cingulate. Together, our findings suggest that HIV-1 Tat and morphine differentially induce depressive-like behaviors associated with increased neuroinflammation, synaptic losses, and immune fatigue within the PFC.

Vitamin D3 Supplementation Promotes Regulatory T-Cells to Maintain Immune Homeostasis After Surgery for Early Stages of Colorectal Cancer.

BACKGROUND/AIM: Vitamin D3 (VD3) affects the regulation of the immune system, including the differentiation and function of regulatory T-cells (Tregs). Tregs play an important role in maintaining immune homeostasis in patients with colorectal cancer (CRC). The effects of VD3 on Treg-associated immune function were investigated in Thai patients in the early stages of CRC. MATERIALS AND METHODS: Twenty-eight patients were randomized to one of two groups: Untreated or treatment with VD3 for 3 months. Whole blood samples were collected at baseline, and at 1 and 3 months. Peripheral blood mononuclear cells were isolated and the populations of forkhead box P3-positive Treg cells was analyzed by flow cytometry. The levels of Treg-associated cytokines, interleukin 10 (IL-10) and transforming growth factor beta 1 (TGF-ß1), were measured by enzyme-linked immunosorbent assays. RESULTS: Serum VD3 levels of the VD3-treated group were significantly increased at 1 (p=0.017) and 3 months (p<0.001) compared to the untreated control group. The mean percentage of Tregs was maintained between 1 and 3 months in the VD3-treated group. At 3 months, the untreated group had significantly lower Treg levels than the VD3-treated group (p=0.043). Serum IL-10 levels of the VD3-treated group were statistically increased at 1 month compared to the control group (p=0.032). No significant difference in serum TGF-ß1 levels was observed between the two groups. However, the TGF-ß1 level in the VD3-treated group at 1 month was lower than that of the control. CONCLUSION: Our findings suggest that VD3 supplementation can maintain immune responses in the early stages of CRC, helping to control Treg function. Therefore, VD3 should be supplemented to maintain immune homeostasis, especially in patients with vitamin D deficiency.

Four-Parameter FluoroSpot Assay Reveals That the Varicella Zoster Virus Elicits a Robust Memory T Cell IL-10 Response throughout Childhood.

During childhood, the composition and function of the T cell compartment undergoes significant changes. In healthy individuals, primary infection with herpesviruses is followed by latency, and occasional subclinical reactivation ensures transmission and contributes to an emerging pool of memory T cells. In immunocompromised individuals, herpesviruses can be life threatening. However, knowledge about the spectrum of virus-specific cytokine responses is limited. Here, we investigated peripheral blood mononuclear cells (PBMCs) from children with differential carrier statuses for cytomegalovirus (CMV), Epstein-Barr virus (EBV), and varicella zoster virus (VZV) (n = 32, age 1 to 17 years). We examined memory T cell subsets as well as IFN-γ-, IL-10-, IL-17A-, and IL-22-producing T cells after polyclonal activation or stimulation with viral peptides using flow cytometry and a 4-parameter FluoroSpot assay. Age and herpesvirus carriage influenced the size of the memory T cell subsets. A positive association between age and the number of IFN-γ-, IL-17A- and IL-22-producing T cells was found following polyclonal activation. For CMV, age was positively associated with IL-17A spot-forming cells (SFC), while for VZV, age was negatively associated with IL-22 and positively associated with IFN-γ SFC. Upon activation with CMV, VZV, and EBV peptides, IFN-γ SFCs dominated. Notably, VZV responses were characterized by a higher IL-10 SFC population compared to both CMV and EBV. Our findings suggest that cytokine responses vary across herpesvirus-type-specific memory T cells and may more adequately reflect their composition. An observed deviation between polyclonal and herpesvirus-specific T cell cytokine responses in children needs to be considered when interpreting the associations between herpesvirus carrier status and bulk T cell reactivity. In summary, these findings may have implications for the treatment of immunocompromised patients. IMPORTANCE Infection with herpesviruses accounts for 35 to 40 billion human cases worldwide. Despite this, little is known about how herpesviruses shape the immune system in the asymptomatic carrier. Particularly in children, primary infection is connected to no or mild symptoms ahead of latency for life. Most research on cellular responses against herpesviruses focuses on inflammatory cytokines associated with antiproliferative and antitumor mechanisms and not the spectrum of cytokine responses in healthy humans. This study investigated four divergent cytokine-producing T cell responses to herpesviruses, reflecting different immunological functions. Three common childhood herpesviruses were selected: Epstein-Barr virus, cytomegalovirus, and varicella-zoster virus. Curiously, not all viruses induced the same pattern of cytokines. Varicella-zoster responses were characterized by IL-10, which is considered regulatory. Besides broadening understanding of responses to herpesviruses, our results raise the possibility that reactivation of varicella-zoster may be counterproductive in cancer treatment through the action of IL-10-producing T-cells.

Restricted Recruitment of NK Cells with Impaired Function Is Caused by HPV-Driven Immunosuppressive Microenvironment of Papillomas in Aggressive Juvenile-Onset Recurrent Respiratory Papillomatosis Patients.

Laryngopharynx epithelium neoplasia induced by HPV6/11 infection in juvenile-onset recurrent respiratory papillomatosis (JO-RRP) causes a great health issue characteristic of frequent relapse and aggressive disease progression. Local cell-mediated immunity shaped by the recruitment and activation of cytotoxic effector cells is critical for viral clearance. In this study, we found that NK cells in the papillomas of aggressive JO-RRP patients, in contrast to massive infiltrated T cells, were scarce in number and impaired in activation and cytotoxicity as they were in peripheral blood. Data from cell infiltration analysis indicated that the migration of NK cell to papilloma was restricted in aggressive JO-RRP patients. Further study showed that the skewed chemokine expression in the papillomas and elevated ICAM-1 expression in hyperplastic epithelia cells favored the T cell but not NK cell recruitment in aggressive JO-RRP patients. In parallel to the increased CD3+ T cells, we observed a dramatical increase in Tregs and Treg-promoting cytokines such as IL-4, IL-10 and TGFß in papillomas of aggressive JO-RRP patients. Our study suggested that likely initialized by the intrinsic change in neoplastic epithelial cells with persistent HPV infection, the aggressive papillomas built an entry barrier for NK cell infiltration and formed an immunosuppressive clump to fend off the immune attack from intra-papillomas NK cells. IMPORTANCE Frequent relapse and aggressive disease progression of juvenile-onset recurrent respiratory papillomatosis (JO-RRP) pose a great challenge to the complete remission of HPV 6/11 related laryngeal neoplasia. Local immune responses in papillomas are more relevant to the disease control considering the locale infected restriction of HPV virus in epitheliums. In our study, the restricted NK cell number and reduced expression of activating NKp30 receptor suggested one possible mechanism underlying impaired NK cell defense ability in aggressive JO-RRP papillomas. Meanwhile, the negative impact of HPV persistent infection on NK cell number and function represented yet another example of a chronic pathogen subverting NK cell behavior, affirming a potentially important role for NK cells in viral containment. Further, the skewed chemokine/cytokine expression in the papillomas and the elevated adhesion molecules expression in hyperplastic epithelia cells provided important clues for understanding blocked infiltration and antiviral dysfunction of NK cells in papilloma.

Differential genotypes of TNF-α and IL-10 for immunological diagnosis in discoid lupus erythematosus and oral lichen planus: A narrative review.

Discoid lupus erythematosus and oral lichen planus are chronic systemic immune system-mediated diseases with unclear etiology and pathogenesis. The oral mucosa is the common primary site of pathogenesis in both, whereby innate and adaptive immunity and inflammation play crucial roles. The clinical manifestations of discoid lupus erythematosus on the oral mucosa are very similar to those of oral lichen planus; therefore, its oral lesion is classified under oral lichenoid lesions. In practice, the differential diagnosis of discoid lupus erythematosus and oral lichen planus has always relied on the clinical manifestations, with histopathological examination as an auxiliary diagnostic tool. However, the close resemblance of the clinical manifestations and histopathology proves challenging for accurate differential diagnosis and further treatment. In most cases, dentists and pathologists fail to distinguish between the conditions during the early stages of the lesions. It should be noted that both are considered to be precancerous conditions, highlighting the significance of early diagnosis and treatment. In the context of unknown etiology and pathogenesis, we suggest a serological and genetic diagnostic method based on TNF-α and IL-10. These are the two most common cytokines produced by the innate and adaptive immune systems and they play a fundamental role in maintaining immune homeostasis and modulating inflammation. The prominent variability in their expression levels and gene polymorphism typing in different lesions compensates for the low specificity of current conventional diagnostic protocols. This new diagnostic scheme, starting from the immunity and inflammation of the oral mucosa, enables simultaneous comparison of discoid lupus erythematosus and oral lichen planus. With relevant supportive evidence, this information can enhance physicians' understanding of the two diseases, contribute to precision medicine, and aid in prevention of precancerous conditions.

Exosomes with FOXP3 from gene-modified dendritic cells ameliorate the development of EAE by regulating the balance of Th/Treg.

Objective: To investigate the efficiency and potential mechanisms of exosomes from dendritic cells (DCs) transfected with Forkhead box protein P3 (FOXP3) in the development of experimental autoimmune encephalomyelitis (EAE). Method: Mouse bone marrow-derived immature DCs were loaded with adenovirus carrying FOXP3 gene, and exosomes were generated. Then the exosomes with FOXP3 (FOXP3-EXOs) were co-cultured with CD4+T cell in vitro to evaluate their potential on CD4+T cell proliferation and differentiation, and injected into EAE mice to assess their effects on the development of EAE. Result: FOXP3-EXOs were effective to inhibit the CD4+T cell proliferation and the production of Interferon gamma (IFN-γ), interleukin (IL)-6, and IL-17, while they promoted the production of IL-10 in vitro. Moreover, FOXP3-EXOs treatment significantly decreased the neurological scores, reduced the infiltration of inflammatory cells into the spinal cord, and decreased demyelination in comparison to saline and Con-EXOs treated EAE mice. Moreover, the FOXP3-EXOs treatment resulted in obvious increases in the levels of regulatory T (Treg) cells and IL-10, whereas levels of T helper 1 (Th1) cells, Th17 cells, IFN-γ, IL-6, and IL-17 decreased significantly in the splenocyte culture of EAE mice. Conclusion: The present study preliminarily investigated the effects and potential mechanisms of FOXP3-EXOs in EAE and revealed that the FOXP3-EXOs could inhibit the production of Th1 and Th17 cells and promote the production of Treg cells as well as ameliorate the development of EAE. The neuroprotective effects of FOXP3-EXOs on EAE are likely due to the regulation of Th/Treg balance.

Adipose­derived mesenchymal stem cell­derived HCAR1 regulates immune response in the attenuation of sepsis.

Sepsis serves as a leading cause of admission to and death of patients in the intensive care unit (ICU) and is described as a systemic inflammatory response syndrome caused by abnormal host response to infection. Adipose­derived mesenchymal stem cells (ADSCs) have exhibited reliable and promising clinical application potential in multiple disorders. However, the function and the mechanism of ADSCs in sepsis remain elusive. In the present study, the crucial inhibitory effect of ADSC­derived hydroxy­carboxylic acid receptor 1 (HCAR1) on sepsis was identified. Reverse transcription quantitative­PCR determined that the mRNA expression of HCAR1 was reduced while the mRNA expression of Toll­like receptor 4 (TLR4), major histocompatibility complex class II (MHC II), NOD­like receptor family pyrin domain containing 3 (NLRP3), and the levels of interleukin­1ß (IL­1ß), tumor necrosis factor­α (TNF­α), interleukin­10 (IL­10), and interleukin­18 (IL­18) were enhanced in the peripheral blood of patients with sepsis. The expression of HCAR1 was negatively correlated with TLR4 (r=­0.666), MHC II (r=­0.587), and NLRP3 (r=­0.621) expression and the expression of TLR4 was positively correlated with NLRP3 (r=0.641), IL­1ß (r=0.666), TNF­α (r=0.606), and IL­18 (r=0.624) levels in the samples. Receiver operating characteristic (ROC) curve analysis revealed that the area under the ROC curve (AUC) of HCAR1, TLR4, MHC II and NLRP3 mRNA expression was 0.830, 0.853, 0.735 and 0.945, respectively, in which NLRP3 exhibited the highest diagnostic value, and the AUC values of IL­1ß, IL­18, TNF­α, and IL­10 were 0.751, 0.841, 0.924 and 0.729, respectively, in which TNF­α exhibited the highest diagnostic value. A sepsis rat model was established by injecting lipopolysaccharide (LPS) and the rats were randomly divided into 5 groups, including a normal control group (NC group; n=6), a sepsis model group (LPS group; n=6), an ADSC transplantation group (L + M group; n=6), a combined HCAR1 receptor agonist group [L + HCAR1 inducer (Gi) + M group; n=6], and a combined HCAR1 receptor inhibitor group [L + HCAR1 blocker (Gk) + M group; n=6]. Hematoxylin and eosin staining determined that ADSCs attenuated the lung injury of septic rats and ADSC­derived HCAR1 enhanced the effect of ADSCs. The expression of HCAR1, TLR4, MHC II, NLRP3, IL­1ß, IL­18 and TNF­α levels were suppressed by ADSCs and the effect was further induced by ADSC­derived HCAR1. However, ADSC­derived HCAR1 induced the levels of anti­inflammatory factor IL­10. The negative correlation of HCAR1 expression with TLR4, MHC II, and NLRP3 expression in the peripheral blood and lung tissues of the rats was then identified. It is thus concluded that ADSC­derived HCAR1 regulates immune response in the attenuation of sepsis. ADSC­derived HCAR1 may be a promising therapeutic strategy for sepsis.

Deciphering the balance of IL-6/IL-10 cytokines in severe to critical COVID-19 patients.

The severity of COVID-19 is largely determined by the inflammatory response, a "Cytokine storm," that involves both pro- and anti-inflammatory cytokines. In the current study we investigated the balance of pro- and anti-inflammatory status as represented by the levels of IL-6/IL-10 in severe to critical COVID-19 patients. 66 confirmed COVID-19 patients admitted to the ICU were categorized into groups according to the mortality and respiratory failure. Data were collected retrospectively in ICU, including a peripheral immune cells and infection-related biomarker CRP. The measurements of cytokine levels were performed by Immulite analyzer for IL-6 and ELISA sandwich for IL-10. In addition, longitudinal measurement of IL-6 was performed during 5 days post admission. Longitudinal assays showed that IL-6 was sustained at a medium level within 5 days post admission in severe cases who survived or not requiring mechanical ventilation, whereas it was sustained at high levels throughout the disease course in either deceased cases or who developed respiratory failure. The ratio of IL-6/lymphocytes was positively correlated with the risk of mortality, while IL-10/lymphocytes ratio could predict respiratory failure in ICU. IL-6/IL-10 profiling revealed that deceased patients have different magnitudes of both IL-6 and IL-10 cytokine release. Notably, excessive levels of IL-6 concomitant with high levels of IL-10 were more common in diseased COVID-19 patients. Taking into account the IL-6/IL-10 profiling may help clinicians to identify the right time of anti-inflammation treatment and select patients who will respond to anti-cytokine therapies and maintain an adequate inflammatory response for SARS-CoV-2 clearance.

Antiviral Toll-like Receptor Signaling in Non-Parenchymal Liver Cells Is Restricted to TLR3.

The role of non-parenchymal liver cells as part of the hepatic, innate immune system in the defense against hepatotropic viruses is not well understood. Here, primary human Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells were isolated from liver tissue obtained after tumor resections or liver transplantations. Cells were stimulated with Toll-like receptor 1-9 ligands for 6-24 h. Non-parenchymal liver cells expressed and secreted inflammatory cytokines (IL6, TNF and IL10). Toll-like receptor- and cell type-specific downstream signals included the phosphorylation of NF-κB, AKT, JNK, p38 and ERK1/2. However, only supernatants of TLR3-activated Kupffer cells, liver sinusoidal endothelial cells and hepatic stellate cells contained type I and type III interferons and mediated an antiviral activity in the interferon-sensitive subgenomic hepatitis C virus replicon system. The antiviral effect could not be neutralized by antibodies against IFNA, IFNB nor IFNL, but could be abrogated using an interferon alpha receptor 2-specific neutralization. Interestingly, TLR3 responsiveness was enhanced in liver sinusoidal endothelial cells isolated from hepatitis C virus-positive donors, compared to uninfected controls. In conclusion, non-parenchymal liver cells are potent activators of the hepatic immune system by mediating inflammatory responses. Furthermore, liver sinusoidal endothelial cells were identified to be hyperresponsive to viral stimuli in chronic hepatitis C virus infection.

Engineering immunomodulatory and osteoinductive implant surfaces via mussel adhesion-mediated ion coordination and molecular clicking.

Immune response and new tissue formation are important aspects of tissue repair. However, only a single aspect is generally considered in previous biomedical interventions, and the synergistic effect is unclear. Here, a dual-effect coating with immobilized immunomodulatory metal ions (e.g., Zn2+) and osteoinductive growth factors (e.g., BMP-2 peptide) is designed via mussel adhesion-mediated ion coordination and molecular clicking strategy. Compared to the bare TiO2 group, Zn2+ can increase M2 macrophage recruitment by up to 92.5% in vivo and upregulate the expression of M2 cytokine IL-10 by 84.5%; while the dual-effect of Zn2+ and BMP-2 peptide can increase M2 macrophages recruitment by up to 124.7% in vivo and upregulate the expression of M2 cytokine IL-10 by 171%. These benefits eventually significantly enhance bone-implant mechanical fixation (203.3 N) and new bone ingrowth (82.1%) compared to the bare TiO2 (98.6 N and 45.1%, respectively). Taken together, the dual-effect coating can be utilized to synergistically modulate the osteoimmune microenvironment at the bone-implant interface, enhancing bone regeneration for successful implantation.

TNF Receptor-Associated Factor 5 Limits IL-27 Receptor Signaling in CD4+ T Lymphocytes.

TNF receptor-associated factor 5 (TRAF5) restrains early signaling activity of the IL-6 receptor in naive CD4+ T cells by interacting with the shared gp130 chain, although TRAF5 was initially discovered as a cytoplasmic adaptor protein to activate signaling mediated by TNF receptor family molecules. This leads to the question of whether TRAF5 limits signaling via the receptor for IL-27, which is composed of gp130 and WSX-1. The aim of this study is to clarify the role of TRAF5 in IL-27 receptor signaling and to understand the differential role of TRAF5 on cytokine receptor signaling. We found that Traf5 -/- CD4+ T cells displayed significantly higher levels of phosphorylated STAT1 and STAT-regulated genes Socs3 and Tbx21, as early as 1 h after IL-27 exposure when compared with Traf5 +/+ CD4+ T cells. Upon IL-27 and TCR signals, the Traf5 deficiency significantly increased the induction of IL-10 and promoted the proliferation of CD4+ T cells. Traf5 -/- mice injected with IL-27 displayed significantly enhanced delayed-type hypersensitivity responses, demonstrating that TRAF5 works as a negative regulator for IL-27 receptor signaling. In contrast, IL-2 and proliferation mediated by glucocorticoid-induced TNF receptor-related protein (GITR) and TCR signals were significantly decreased in Traf5 -/- CD4+ T cells, confirming that TRAF5 works as a positive regulator for cosignaling via GITR. Collectively, our results demonstrate that TRAF5 reciprocally controls signals mediated by the IL-27 receptor and GITR in CD4+ T cells and suggest that the regulatory activity of TRAF5 in gp130 is distinct from that in TNF receptor family molecules in a T cell.

Functional Effects of Cardiomyocyte Injury in COVID-19.

COVID-19 affects multiple organs. Clinical data from the Mount Sinai Health System show that substantial numbers of COVID-19 patients without prior heart disease develop cardiac dysfunction. How COVID-19 patients develop cardiac disease is not known. We integrated cell biological and physiological analyses of human cardiomyocytes differentiated from human induced pluripotent stem cells (hiPSCs) infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the presence of interleukins (ILs) with clinical findings related to laboratory values in COVID-19 patients to identify plausible mechanisms of cardiac disease in COVID-19 patients. We infected hiPSC-derived cardiomyocytes from healthy human subjects with SARS-CoV-2 in the absence and presence of IL-6 and IL-1ß. Infection resulted in increased numbers of multinucleated cells. Interleukin treatment and infection resulted in disorganization of myofibrils, extracellular release of troponin I, and reduced and erratic beating. Infection resulted in decreased expression of mRNA encoding key proteins of the cardiomyocyte contractile apparatus. Although interleukins did not increase the extent of infection, they increased the contractile dysfunction associated with viral infection of cardiomyocytes, resulting in cessation of beating. Clinical data from hospitalized patients from the Mount Sinai Health System show that a significant portion of COVID-19 patients without history of heart disease have elevated troponin and interleukin levels. A substantial subset of these patients showed reduced left ventricular function by echocardiography. Our laboratory observations, combined with the clinical data, indicate that direct effects on cardiomyocytes by interleukins and SARS-CoV-2 infection might underlie heart disease in COVID-19 patients. IMPORTANCE SARS-CoV-2 infects multiple organs, including the heart. Analyses of hospitalized patients show that a substantial number without prior indication of heart disease or comorbidities show significant injury to heart tissue, assessed by increased levels of troponin in blood. We studied the cell biological and physiological effects of virus infection of healthy human iPSC-derived cardiomyocytes in culture. Virus infection with interleukins disorganizes myofibrils, increases cell size and the numbers of multinucleated cells, and suppresses the expression of proteins of the contractile apparatus. Viral infection of cardiomyocytes in culture triggers release of troponin similar to elevation in levels of COVID-19 patients with heart disease. Viral infection in the presence of interleukins slows down and desynchronizes the beating of cardiomyocytes in culture. The cell-level physiological changes are similar to decreases in left ventricular ejection seen in imaging of patients' hearts. These observations suggest that direct injury to heart tissue by virus can be one underlying cause of heart disease in COVID-19.