CXCL1 promotes immune escape in colorectal cancer by autophagy-mediated MHC-I degradation.

BACKGROUND: Immunotherapy is now seen as a potential remedy for colorectal cancer (CRC). Chemokines play a crucial role in tumors, including CRC, which contains CXCL1. We attempted to study how CXCL1 impacts immune escape in CRC. METHODS: Bioinformatics analysis was used to examine CXCL1 level in CRC. qRT-PCR was used to assess CXCL1 and MHC-I (HLA-A, B, C) levels. Cell Counting Kit-8 (CCK-8) was used to measure cell viability. Cytotoxicity assay kit was utilized to assay CD8+ T cell cytotoxicity against CRC. Flow cytometry tested proliferation and apoptosis of CD8+ T cells. Chemotaxis assay evaluated chemotaxis of CD8+ T cells towards CRC. Immunofluorescence examined expression of autophagy marker LC3 and localization of NBR1/MHC-I. Western blot analysis measured protein levels of chemokines CXCL9 and CXCL10, autophagy-related proteins LC3-I and LC3-II, and MHC-I (HLA-A, B, C). RESULTS: Bioinformatics analysis and qRT-PCR presented that CXCL1 was upregulated in CRC. Cell experiments demonstrated that CXCL1 overexpression promoted immune escape in CRC. Rescue experiments revealed that the autophagy inducer Rapa could attenuate the inhibitory effect of CXCL1 low expression on immune escape in CRC. Further studies showed that CXCL1 promoted immune escape in CRC by autophagy-mediated MHC-I degradation. CONCLUSION: CXCL1 promoted immune escape in CRC by autophagy-mediated MHC-I degradation, suggesting that CXCL1 may be a possible immunotherapeutic target for CRC.

The chemerin/CMKLR1 axis regulates intestinal graft-versus-host disease.

Gastrointestinal graft-versus-host disease (GvHD) is a major cause of mortality and morbidity following allogeneic bone marrow transplantation (allo-BMT). Chemerin is a chemotactic protein that recruits leukocytes to inflamed tissues by interacting with ChemR23/CMKLR1, a chemotactic receptor expressed by leukocytes, including macrophages. During acute GvHD, chemerin plasma levels were strongly increased in allo-BM-transplanted mice. The role of the chemerin/CMKLR1 axis in GvHD was investigated using Cmklr1-KO mice. WT mice transplanted with an allogeneic graft from Cmklr1-KO donors (t-KO) had worse survival and more severe GvHD. Histological analysis demonstrated that the gastrointestinal tract was the organ mostly affected by GvHD in t-KO mice. The severe colitis of t-KO mice was characterized by massive neutrophil infiltration and tissue damage associated with bacterial translocation and exacerbated inflammation. Similarly, Cmklr1-KO recipient mice showed increased intestinal pathology in both allogeneic transplant and dextran sulfate sodium-induced colitis. Notably, the adoptive transfer of WT monocytes into t-KO mice mitigated GvHD manifestations by decreasing gut inflammation and T cell activation. In patients, higher chemerin serum levels were predictive of GvHD development. Overall, these results suggest that CMKLR1/chemerin may be a protective pathway for the control of intestinal inflammation and tissue damage in GvHD.

Neural-Cell-Intrinsic NF-κB Signaling Enhances Reovirus Virulence.

Pathological effects of apoptosis associated with viral infections of the central nervous system are an important cause of morbidity and mortality. Reovirus is a neurotropic virus that causes apoptosis in neurons, leading to lethal encephalitis in newborn mice. Reovirus-induced encephalitis is diminished in mice with germ line ablation of NF-κB subunit p50. It is not known whether the proapoptotic function of NF-κB is mediated by neural-cell-intrinsic (neural-intrinsic) processes, NF-κB-regulated cytokine production by inflammatory cells, or a combination of both. To determine the contribution of cell type-specific NF-κB signaling in reovirus-induced neuronal injury, we established mice that lack NF-κB p65 expression in neural cells using the Cre/loxP recombination system. Following intracranial inoculation of reovirus, 50% of wild-type (WT) mice succumbed to infection, whereas more than 90% of mice lacking neural cell NF-κB p65 (Nsp65-/-) survived. While viral loads in brains of WT and Nsp65-/- mice were comparable, histological analysis revealed that reovirus antigen-positive areas in the brains of WT mice displayed increased immunoreactivity for cleaved caspase-3, a marker of apoptosis, relative to Nsp65-/- mice. These data suggest that neural-intrinsic NF-κB-dependent factors are essential mediators of reovirus neurovirulence. RNA sequencing analysis of reovirus-infected brain cortices of WT and Nsp65-/- mice suggests that NF-κB activation in neuronal cells upregulates genes involved in innate immunity, inflammation, and cell death following reovirus infection. A better understanding of the contribution of cell type-specific NF-κB-dependent signaling to viral neuropathogenesis could inform development of new therapeutics that target and protect highly vulnerable cell populations. IMPORTANCE Viral encephalitis contributes to illness and death in children and adults worldwide and has limited treatment options. Identifying common host factors upregulated by neurotropic viruses can enhance an understanding of virus-induced neuropathogenesis and aid in development of therapeutics. Although many neurotropic viruses activate NF-κB during infection, mechanisms by which NF-κB regulates viral neuropathogenesis and contributes to viral encephalitis are not well understood. We established mice in which NF-κB expression is ablated in neural tissue to study the function of NF-κB in reovirus neurovirulence and identify genes activated by NF-κB in response to reovirus infection in the central nervous system. Encephalitis following reovirus infection was dampened in mice lacking neural cell NF-κB. Reovirus induced a chemokine profile in the brain that was dependent on NF-κB signaling and was similar to chemokine profiles elicited by other neurotropic viruses. These data suggest common underlying mechanisms of encephalitis caused by neurotropic viruses and potentially shared therapeutic targets.

The value of thymus and activation related chemokine immunohistochemistry in classic Hodgkin lymphoma diagnostics.

AIMS: Classic Hodgkin lymphoma (cHL) should be distinguished from its wide variety of histological mimics, including reactive conditions and mature B and T cell neoplasms. Thymus and activation-related chemokine (TARC) is produced in extremely high quantities by the Hodgkin/Reed-Sternberg (HRS) tumour cells and is largely responsible for the attraction of CD4+ T cells into the cHL tumour micro-environment. In the current study we evaluated the diagnostic potential of TARC immunohistochemistry in daily practice in a tertiary referral centre in the Netherlands. METHODS AND RESULTS: A total of 383 cases, approximately half of which were cHL mimics, were prospectively evaluated in the period from June 2014 to November 2020. In 190 cHL cases, 92% were TARC-positive and the majority of cases showed strong and highly specific staining in all HRS cells (77%). In most cases, TARC could discriminate between nodular lymphocyte-predominant and lymphocyte-rich Hodgkin lymphoma. HRS-like cells in mature lymphoid neoplasms were rarely positive (6.4%) and there was no TARC staining at all in 64 reactive lymphadenopathies. CONCLUSIONS: TARC immunohistochemistry has great value in differentiating between cHL and its mimics, including nodular lymphocyte-predominant Hodgkin lymphoma, reactive lymphadenopathies and mature lymphoid neoplasms with HRS-like cells.

IL-17D affects the chemokines and chemokine receptors of intestinal epithelial cells under hyperoxia.

IL-17D is a new member of the IL-17 family. Currently, it is believed that IL-17D can directly act on immune cells or may indirectly modulate immune responses by regulating cytokine expression. Herein, we hypothesized that IL-17D regulates the expression of chemokines in intestinal epithelial cells, in turn modulating the immune response within intestinal mucosa under hyperoxia. To explore this notion, newborn rats were divided into a hyperoxia group (85 % O2) and control group (21 % O2). Small intestinal tissues were obtained from neonatal rats at 3, 7, 10, and 14 days. Similarly, intestinal epithelial cells were treated by hyperoxia (85 % O2) as the hyperoxia group or were incubated under normal oxygen (21 % O2) as the control group. Finally, intestinal epithelial cells subjected to hyperoxia were treated with recombinant IL-17D and IL-17D antibodies for 24, 48, and 72 h. Immunohistochemistry, western blot, and reverse transcription-quantitative polymerase chain reaction were used to detect the expression levels of chemokines and chemokine receptors in intestinal tissues of newborn rats and intestinal epithelial cells. We found that hyperoxia affected chemokine expression both in vivo and in vitro. Under hyperoxia, IL-17D promoted the expression of CCL2, CCL25, CCL28, and CCR9 in intestinal epithelial cells while downregulating CCR2, CCR5, CCL5, and CCL20. Our findings provide a basis for further study on the effects of hyperoxia-induced intestinal inflammation and intestinal injury.

Regulation of pancreatic cancer therapy resistance by chemokines.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by high resistance and poor response to chemotherapy. In addition, the poorly immunogenic pancreatic tumors constitute an immunosuppressive tumor microenvironment (TME) that render immunotherapy-based approaches ineffective. Understanding the mechanisms of therapy resistance, identifying new targets, and developing effective strategies to overcome resistance can significantly impact the management of PDAC patients. Chemokines are small soluble factors that are significantly deregulated during PDAC pathogenesis, contributing to tumor growth, metastasis, immune cell trafficking, and therapy resistance. Thus far, different chemokine pathways have been explored as therapeutic targets in PDAC, with some promising results in recent clinical trials. Particularly, immunotherapies such as immune check point blockade therapies and CAR-T cell therapies have shown promising results when combined with chemokine targeted therapies. Considering the emerging pathological and clinical significance of chemokines in PDAC, we reviewed major chemokine-regulated pathways leading to therapy resistance and the ongoing endeavors to target chemokine signaling in PDAC. This review discusses the role of chemokines in regulating therapy resistance in PDAC and highlights the continuing efforts to target chemokine-regulated pathways to improve the efficacy of various treatment modalities.

Lymph-derived chemokines direct early neutrophil infiltration in the lymph nodes upon Staphylococcus aureus skin infection.

A large number of neutrophils infiltrate the lymph node (LN) within 4 h after Staphylococcus aureus skin infection (4 h postinfection [hpi]) and prevent systemic S. aureus dissemination. It is not clear how infection in the skin can remotely and effectively recruit neutrophils to the LN. Here, we found that lymphatic vessel occlusion substantially reduced neutrophil recruitment to the LN. Lymphatic vessels effectively transported bacteria and proinflammatory chemokines (i.e., Chemokine [C-X-C motif] motif 1 [CXCL1] and CXCL2) to the LN. However, in the absence of lymph flow, S. aureus alone in the LN was insufficient to recruit neutrophils to the LN at 4 hpi. Instead, lymph flow facilitated the earliest neutrophil recruitment to the LN by delivering chemokines (i.e., CXCL1, CXCL2) from the site of infection. Lymphatic dysfunction is often found during inflammation. During oxazolone (OX)-induced skin inflammation, CXCL1/2 in the LN was reduced after infection. The interrupted LN conduits further disrupted the flow of lymph and impeded its communication with high endothelial venules (HEVs), resulting in impaired neutrophil migration. The impaired neutrophil interaction with bacteria contributed to persistent infection in the LN. Our studies showed that both the flow of lymph from lymphatic vessels to the LN and the distribution of lymph in the LN are critical to ensure optimal neutrophil migration and timely innate immune protection in S. aureus infection.

The primary macrophage chemokine, CCL2, is not necessary after a peripheral nerve injury for macrophage recruitment and activation or for conditioning lesion enhanced peripheral regeneration.

BACKGROUND: Peripheral nerve injuries stimulate the regenerative capacity of injured neurons through a neuroimmune phenomenon termed the conditioning lesion (CL) response. This response depends on macrophage accumulation in affected dorsal root ganglia (DRGs) and peripheral nerves. The macrophage chemokine CCL2 is upregulated after injury and is allegedly required for stimulating macrophage recruitment and pro-regenerative signaling through its receptor, CCR2. In these tissues, CCL2 is putatively produced by neurons in the DRG and Schwann cells in the distal nerve. METHODS: Ccl2fl/fl mice were crossed with Advillin-Cre, P0-Cre, or both to create conditional Ccl2 knockouts (CKOs) in sensory neurons, Schwann cells, or both to hypothetically remove CCL2 and macrophages from DRGs, nerves or both. CCL2 was localized using Ccl2-RFPfl/fl mice. CCL2-CCR2 signaling was further examined using global Ccl2 KOs and Ccr2gfp knock-in/knock-outs. Unilateral sciatic nerve transection was used as the injury model, and at various timepoints, chemokine expression, macrophage accumulation and function, and in vivo regeneration were examined using qPCR, immunohistochemistry, and luxol fast blue staining. RESULTS: Surprisingly, in all CKOs, DRG Ccl2 gene expression was decreased, while nerve Ccl2 was not. CCL2-RFP reporter mice revealed CCL2 expression in several cell types beyond the expected neurons and Schwann cells. Furthermore, macrophage accumulation, myelin clearance, and in vivo regeneration were unaffected in all CKOs, suggesting CCL2 may not be necessary for the CL response. Indeed, Ccl2 global knockout mice showed normal macrophage accumulation, myelin clearance, and in vivo regeneration, indicating these responses do not require CCL2. CCR2 ligands, Ccl7 and Ccl12, were upregulated after nerve injury and perhaps could compensate for the absence of Ccl2. Finally, Ccr2gfp knock-in/knock-out animals were used to differentiate resident and recruited macrophages in the injured tissues. Ccr2gfp/gfp KOs showed a 50% decrease in macrophages in the distal nerve compared to controls with a relative increase in resident macrophages. In the DRG there was a small but insignificant decrease in macrophages. CONCLUSIONS: CCL2 is not necessary for macrophage accumulation, myelin clearance, and axon regeneration in the peripheral nervous system. Without CCL2, other CCR2 chemokines, resident macrophage proliferation, and CCR2-independent monocyte recruitment can compensate and allow for normal macrophage accumulation.

Brain motor and fear circuits regulate leukocytes during acute stress.

The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.

Identification and validation of EMT-immune-related prognostic biomarkers CDKN2A, CMTM8 and ILK in colon cancer.

Colon cancer (CC) is a disease with high incidence and mortality rate. The interaction between epithelial-mesenchymal transition (EMT) and immune status has important clinical significance. We aim to identify EMT-immune-related prognostic biomarkers in colon cancer. The GEO2R and GEPIA 2.0 were utilized to calculate the differential expression genes between CC and normal mucosa. Immport, InnateDB and EMTome databases were used to define EMT-immune-related genes. We conducted batch prognostic analysis by TCGA data. The expression patterns were verified by multiple datasets and lab experiments. GEPIA 2.0 and TIMER 2.0 were utilized to analyze the correlation of the hub genes with EMT markers and immune infiltration. GeneMANIA, STRING, and Metascape were used for co-expression and pathway enrichment analysis. Finally, we established a signature by the method of multivariate Cox regression analysis. CDKN2A, CMTM8 and ILK were filtered out as prognostic genes. CDKN2A and CMTM8 were up-regulated, while ILK was down-regulated in CC. CDKN2A was positively correlated with infiltration of macrophages, Th2 cells, Treg cells, and negatively correlated with NK cells. CMTM8 was negatively correlated with CD8+ T cells, dendritic cells, and NK cells. ILK was positively correlated with CD8+ T cells and dendritic cells. Moreover, CDKN2A, CMTM8 and ILK were significantly correlated with EMT markers. The three genes could participate in the TGF-ß pathway. The prognosis model established by the three hub genes was an independent prognosis factor, which can better predict the prognosis. CDKN2A, CMTM8 and ILK are promising prognostic biomarkers and may be potential therapeutic targets in colon cancer.

Sensing plasma membrane pore formation induces chemokine production in survivors of regulated necrosis.

Although overwhelming plasma membrane integrity loss leads to cell lysis and necrosis, cells can tolerate a limited level of plasma membrane damage, undergo ESCRT-III-mediated repair, and survive. Here, we find that cells which undergo limited plasma membrane damage from the pore-forming actions of MLKL, GSDMD, perforin, or detergents experience local activation of PKCs through Ca2+ influx at the damage sites. S660-phosphorylated PKCs subsequently activate the TAK1/IKKs axis and RelA/Cux1 complex to trigger chemokine expressions. We observe that in late-stage cancers, cells with active MLKL show expression of CXCL8. Similar expression induction is also found in ischemia-injured kidneys. Chemokines generated in this manner are also indispensable for recruiting immune cells to the dead and dying cells. This plasma membrane integrity-sensing pathway is similar to the well-established yeast cell wall integrity signaling pathway at molecular level, and this suggests an evolutionary conserved mechanism to respond to the cellular barrier damage.

Immune Profiling of COVID-19 in Correlation with SARS and MERS.

Acute respiratory distress syndrome (ARDS) is a major complication of the respiratory illness coronavirus disease 2019, with a death rate reaching up to 40%. The main underlying cause of ARDS is a cytokine storm that results in a dysregulated immune response. This review discusses the role of cytokines and chemokines in SARS-CoV-2 and its predecessors SARS-CoV and MERS-CoV, with particular emphasis on the elevated levels of inflammatory mediators that are shown to be correlated with disease severity. For this purpose, we reviewed and analyzed clinical studies, research articles, and reviews published on PubMed, EMBASE, and Web of Science. This review illustrates the role of the innate and adaptive immune responses in SARS, MERS, and COVID-19 and identifies the general cytokine and chemokine profile in each of the three infections, focusing on the most prominent inflammatory mediators primarily responsible for the COVID-19 pathogenesis. The current treatment protocols or medications in clinical trials were reviewed while focusing on those targeting cytokines and chemokines. Altogether, the identified cytokines and chemokines profiles in SARS-CoV, MERS-CoV, and SARS-CoV-2 provide important information to better understand SARS-CoV-2 pathogenesis and highlight the importance of using prominent inflammatory mediators as markers for disease diagnosis and management. Our findings recommend that the use of immunosuppression cocktails provided to patients should be closely monitored and continuously assessed to maintain the desirable effects of cytokines and chemokines needed to fight the SARS, MERS, and COVID-19. The current gap in evidence is the lack of large clinical trials to determine the optimal and effective dosage and timing for a therapeutic regimen.

Upregulation of IFNÉ£-mediated chemokines dominate the immune transcriptome of muscle-invasive urothelial carcinoma.

Tumor inflammation is prognostically significant in high-grade muscle-invasive bladder cancer (MIBC). However, the underlying mechanisms remain elusive. To identify inflammation-associated immune gene expression patterns, we performed transcriptomic profiling of 40 MIBC archival tumors using the NanoString nCounter Human v.1.1 PanCancer Panel. Findings were validated using the TCGA MIBC dataset. Unsupervised and supervised clustering identified a distinctive immune-related gene expression profile for inflammation, characterized by significant upregulation of 149 genes, particularly chemokines, a subset of which also had potential prognostic utility. Some of the most enriched biological processes were lymphocyte activation and proliferation, leukocyte adhesion and migration, antigen processing and presentation and cellular response to IFN-γ. Upregulation of numerous IFN-γ-inducible chemokines, class II MHC molecules and immune checkpoint genes was detected as part of the complex immune response to MIBC. Further, B-cell markers linked to tertiary lymphoid structures were upregulated, which in turn is predictive of tumor response to immunotherapy and favorable outcome. Our findings of both an overall activated immune profile and immunosuppressive microenvironment provide novel insights into the complex immune milieu of MIBC with inflammation and supports its clinical significance for predicting prognosis and immunotherapeutic responsiveness, which warrants further investigation. This may open novel opportunities to identify mechanisms for developing new immunotherapeutic strategies.

Expression of immune response genes in human corneal epithelial cells interacting with Aspergillus flavus conidia.

BACKGROUND: Aspergillus flavus, one of the causative agents of human fungal keratitis, can be phagocytosed by human corneal epithelial (HCE) cells and the conidia containing phagosomes mature into phagolysosomes. But the immunological responses of human corneal epithelial cells interacting with A. flavus are not clear. In this study, we report the expression of immune response related genes of HCE cells exposed to A. flavus spores using targeted transcriptomics. METHODS: Human corneal epithelial cell line and primary cultures were grown in a six-well plate and used for coculture experiments. Internalization of the conidia was confirmed by immunofluorescence microscopy of the colocalized endosomal markers CD71 and LAMP1. Total RNA was isolated, and the quantity and quality of the isolated RNA were assessed using Qubit and Bioanalyzer. NanoString nCounter platform was used for the analysis of mRNA abundance using the Human Immunology panel. R-package and nSolver software were used for data analysis. KEGG and FunRich 3.1.3 tools were used to analyze the differentially expressed genes. RESULTS: Different morphotypes of conidia were observed after 6 h of coculture with human corneal epithelial cells and found to be internalized by epithelial cells. NanoString profiling showed more than 20 differentially expressed genes in immortalized human corneal epithelial cell line and more than ten differentially expressed genes in primary corneal epithelial cells. Distinct set of genes were altered in their expression in cell line and primary corneal epithelial cells. KEGG pathway analysis revealed that genes associated with TNF signaling, NF-KB signaling, and Th17 signaling were up-regulated, and genes associated with chemokine signaling and B cell receptor signaling were down regulated. FunRich pathway analysis showed that pathways such as CDC42 signaling, PI3K signaling, and Arf6 trafficking events were activated by the clinical isolates CI1123 and CI1698 in both type of cells. CONCLUSIONS: Combining the transcript analysis data from cell lines and primary cultures, we showed the up regulation of immune defense genes in A. flavus infected cells. At the same time, chemokine signaling and B cell signaling pathways are downregulated. The variability in the expression levels in the immortalized cell line and the primary cultures is likely due to the variable epigenetic reprogramming in the immortalized cells and primary cultures in the absence of any changes in the genome. It highlights the importance of using both cell types in host-pathogen interaction studies.

Microglia Do Not Restrict SARS-CoV-2 Replication following Infection of the Central Nervous System of K18-Human ACE2 Transgenic Mice.

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited antiviral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, ∼50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine (Cxcl9, Cxcl10, Ccl2, Ccl5 and Ccl19) and cytokine (Ifn-λ and Tnf-α) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease. IMPORTANCE Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.

Breastfeeding and circulating immunological markers during the first 3 years of life: the DIABIMMUNE study.

AIMS/HYPOTHESIS: Our aim was to study the association between duration of breastfeeding and circulating immunological markers during the first 3 years of life in children with HLA-conferred susceptibility to type 1 diabetes. METHODS: We performed a longitudinal analysis of 38 circulating immunological markers (cytokines, chemokines and growth factors) in serum samples from Finnish (56 individuals, 147 samples), Estonian (56 individuals 148 samples) and Russian Karelian children (62 individuals, 149 samples) at 3, 6, 12, 18, 24 and 36 months of age. We also analysed gut inflammation markers (calprotectin and human ß defensin-2) at 3 (n = 96) and 6 months (n = 153) of age. Comparisons of immunological marker medians were performed between children who were breastfed for 6 months or longer vs children who were breastfed for less than 6 months. RESULTS: Breastfeeding for 6 months or longer vs less than 6 months was associated with lower median of serum immunological markers at 6 months (granulocyte-macrophage colony-stimulating factor [GMCSF], macrophage inflammatory protein [MIP-3α]), 12 months (IFN-α2, vascular endothelial growth factor, GMCSF, IFN-γ, IL-21), 18 months (FGF-2, IFN-α2) and 24 months of age (CCL11 [eotaxin], monocyte chemoattractant protein-1, TGFα, soluble CD40 ligand, IL-13, IL-21, IL-5, MIP-1α) (all p < 0.01) but not at 36 months of age. Breastfeeding was not associated with gut inflammation markers at 3 and 6 months of age. CONCLUSIONS/INTERPRETATION: Children who were breastfed for 6 months or longer had lower medians for 14 immunological markers at one or more age points during the first 2 years of life compared with children who were breastfed for less than 6 months. The clinical meaning of the findings is not clear. However, the present study contributes to the understanding of immunological differences in children that have been breastfed longer, and thus provides a mechanistic suggestion for the previously observed associations between breastfeeding and risk of type 1 diabetes.

Effect of HIV suppression on the cytokine network in blood and seminal plasma.

OBJECTIVE: HIV infection disrupts the cytokine network and this disruption is not completely reversed by antiretroviral therapy (ART). Characterization of cytokine changes in blood and genital secretions is important for understanding HIV pathogenesis and the mechanisms of HIV sexual transmission. Here, we characterized the cytokine network in individuals longitudinally sampled before they began ART and after achieving suppression of HIV RNA. METHODS: We measured concentrations of 34 cytokine/chemokines using multiplex bead-based assay in blood and seminal plasma of 19 men with HIV-1 prior to and after viral suppression. We used Partial Least Squares Discriminant Analysis (PLS-DA) to visualize the difference in cytokine pattern between the time points. Any cytokines with VIP scores exceeding 1 were deemed important in predicting suppression status and were subsequently tested using Wilcoxon Signed Rank Tests. RESULTS: PLS-DA projections in blood were fairly similar before and after viral suppression. In contrast, the difference in PLS-DA projection observed in semen emphasizes that the immunological landscape and immunological needs are very different before and after ART in the male genital compartment. When tested individually, four cytokines were significantly different across time points in semen (MIG, IL-15, IL-7, I-TAC), and two in blood (MIG and IP-10). CONCLUSION: Viral suppression with ART impacts the inflammatory milieu in seminal plasma. In contrast, the overall effect on the network of cytokines in blood was modest but consistent with prior analyses. These results identify specific changes in the cytokine networks in semen and blood as the immune system acclimates to chronic, suppressed HIV infection.

Increased cytokines/chemokines and hyponatremia as a possible cause of clinically mild encephalitis/encephalopathy with a reversible splenial lesion associated with acute focal bacterial nephritis.

OBJECTIVE: Clinically mild encephalitis/encephalopathy with a reversible splenial lesion (MERS), the second most common encephalopathy syndrome in Japan, is most often associated with viral infection. Bacterial MERS has been rarely reported but is mostly associated with acute focal bacterial nephritis (AFBN) for an unknown reason. We examined cytokines and chemokines in four MERS patients with AFBN to determine if they play an important role in the pathogenesis. METHODS: We examined the clinical charts and MRI results in four MERS patients with AFBN, and measured 10 cytokines and chemokines in serum and cerebrospinal fluid in the acute phase. These were analyzed using the Mann-Whitney U test, compared with the control group (cases with a non-inflammatory neurological disease). Longitudinal changes in the serum cytokine and chemokine levels were evaluated in two patients. RESULTS: Hyponatremia was observed in all four patients with MERS associated with AFBN (128-134 mEq/L). CSF analysis revealed increased cytokines/chemokines associated with Th1 (CXCL10, TNF-α, IFN-γ), T reg (IL-10), Th17 (IL-6), and neutrophil (IL-8 and CXCL1). In serum, upregulation was observed in those associated with Th1 (CXCL10, TNF-α, IFN-γ), Th17 (IL-6), and inflammasome (IL-1ß). The increased serum cytokines/chemokines in the acute stage normalized within 2 weeks in patients 1 and 2, so examined, in accordance with their clinical improvement. CONCLUSION: Increased cytokines/chemokines and hyponatremia may be factors that explain why AFBN is likely to cause MERS.

Fermented rice bran supplementation attenuates chronic colitis-associated extraintestinal manifestations in female C57BL/6N mice.

Patients with inflammatory bowel disease (IBD) have higher incidence of extraintestinal manifestations (EIM), including liver disorders, sarcopenia, and neuroinflammation. Fermented rice bran (FRB), generated from rice bran (RB), is rich in bioactive compounds, and exhibits anti-colitis activity. However, its role in EIM prevention is still unclear. Here, for the first time, we investigated whether EIM in female C57Bl/6N mice is attenuated by FRB supplementation. EIM was induced by repeated administration of 1.5% dextran sulfate sodium (DSS) in drinking water (4 d) followed by drinking water (12 d). Mice were divided into 3 groups-control (AIN93M), 10% RB, and 10% FRB. FRB ameliorated relapsing colitis and inflammation in muscle by significantly lowering proinflammatory cytokines Tnf-α and Il-6 in serum and advanced glycation end product-specific receptor (Ager) in serum and muscle when compared with the RB and control groups. As FRB reduced aspartate aminotransferase levels and oxidative stress, it might prevent liver disorders. FRB downregulated proinflammatory cytokine and chemokine transcripts responsible for neuroinflammation in the hippocampus and upregulated mRNA expression of G protein coupled receptors (GPRs), Gpr41 and Gpr43, in small and large intestines, which may explain the FRB-mediated protective mechanism. Hence, FRB can be used as a supplement to prevent IBD-associated EIM.

Basal and IL-1ß enhanced chondrocyte chemotactic activity on monocytes are co-dependent on both IKKα and IKKß NF-κB activating kinases.

IKKα and IKKß are essential kinases for activating NF-κB transcription factors that regulate cellular differentiation and inflammation. By virtue of their small size, chemokines support the crosstalk between cartilage and other joint compartments and contribute to immune cell chemotaxis in osteoarthritis (OA). Here we employed shRNA retroviruses to stably and efficiently ablate the expression of each IKK in primary OA chondrocytes to determine their individual contributions for monocyte chemotaxis in response to chondrocyte conditioned media. Both IKKα and IKKß KDs blunted both the monocyte chemotactic potential and the protein levels of CCL2/MCP-1, the chemokine with the highest concentration and the strongest association with monocyte chemotaxis. These findings were mirrored by gene expression analysis indicating that the lowest levels of CCL2/MCP-1 and other monocyte-active chemokines were in IKKαKD cells under both basal and IL-1ß stimulated conditions. We find that in their response to IL-1ß stimulation IKKαKD primary OA chondrocytes have reduced levels of phosphorylated NFkappaB p65pSer536 and H3pSer10. Confocal microscopy analysis revealed co-localized p65 and H3pSer10 nuclear signals in agreement with our findings that IKKαKD effectively blunts their basal level and IL-1ß dependent increases. Our results suggest that IKKα could be a novel OA disease target.