Chronic Kidney Disease Is Characterized by Expansion of a Distinct Proinflammatory Intermediate Monocyte Subtype and by Increased Monocyte Adhesion to Endothelial Cells.

SIGNIFICANCE STATEMENT: CKD is accompanied by abnormal inflammation, which contributes to progressive loss of functional renal tissue and accelerated cardiovascular disease. Although studies have documented that dysregulation of monocyte maturation and function is associated with CKD and its complications, it is not well characterized. This study reveals that a distinctive human monocyte subtype with high propensity for releasing proinflammatory mediators and activating endothelial cells is increased in adults with CKD compared with adults with high cardiovascular risk and normal kidney function. It also demonstrates that human monocyte adhesion to endothelial layers and responses to specific inflammatory migration signals are enhanced in CKD. These findings offer insights into the mechanisms of CKD-associated intravascular and localized inflammation and may suggest potential targets for therapeutic interventions. BACKGROUND: Cardiovascular disease (CVD) in patients with CKD is associated with increased circulating intermediate monocytes (IMs). Dysregulation of monocyte maturation and function is associated with CKD and its complications, but it is incompletely characterized. METHODS: To explore monocyte repertoire abnormalities in CKD, we studied properties of monocyte subpopulations, including IM subpopulations distinguished by HLA-DR expression level, in individuals with or without CKD. Using flow cytometry, we profiled monocyte populations in blood samples from adults with CKD, healthy volunteers (HVs), and patient controls (PCs) with high CVD risk. Monocyte subpopulations were also derived from single-cell RNA-sequencing profiles of paired blood and biopsy samples from kidney transplant recipients. We quantified intracellular cytokine production, migration, and endothelial adhesion in ex vivo assays of PBMCs. RESULTS: Of four predefined blood monocyte subpopulations, only HLA-DR hi IMs were increased in individuals with CKD compared with HVs and PCs. In HVs and patients with CKD, LPS-stimulated HLA-DR hi IMs isolated from blood produced higher amounts of TNF and IL-1 ß than other monocyte populations. Single-cell analysis revealed four monocyte clusters common to blood and kidneys, including an HLA-DR hi IM-like cluster that was enriched in kidneys versus blood. Migration toward CCL5 and CX3CL1 and adhesion to primary endothelial cell layers were increased in monocyte subpopulations in individuals with CKD compared with HVs. Monocyte adhesion to endothelial cells was partly dependent on CX3CR1/CX3CL1 interaction. CONCLUSIONS: CKD is associated with an increased number of a distinctive proinflammatory IM subpopulation and abnormalities of monocyte migration and endothelial adhesion. Dysregulated monocyte maturation and function may represent targetable factors contributing to accelerated CVD in CKD.

Comparison of Single and Repeated Dosing of Anti-Inflammatory Human Umbilical Cord Mesenchymal Stromal Cells in a Mouse Model of Polymicrobial Sepsis.

Mesenchymal stromal cells (MSCs) ameliorate pre-clinical sepsis and sepsis-associated acute kidney injury (SA-AKI) but clinical trials of single-dose MSCs have not indicated robust efficacy. This study investigated immunomodulatory effects of a novel MSC product (CD362-selected human umbilical cord-derived MSCs [hUC-MSCs]) in mouse endotoxemia and polymicrobial sepsis models. Initially, mice received intra-peritoneal (i.p.) lipopolysaccharide (LPS) followed by single i.p. doses of hUC-MSCs or vehicle. Next, mice underwent cecal ligation and puncture (CLP) followed by intravenous (i.v.) doses of hUC-MSCs at 4 h or 4 and 28 h. Analyses included serum/plasma assays of biochemical indices, inflammatory mediators and the AKI biomarker NGAL; multi-color flow cytometry of peritoneal macrophages (LPS) and intra-renal immune cell subpopulations (CLP) and histology/immunohistochemistry of kidney (CLP). At 72 h post-LPS injections, hUC-MSCs reduced serum inflammatory mediators and peritoneal macrophage M1/M2 ratio. Repeated, but not single, hUC-MSC doses administered at 48 h post-CLP resulted in lower serum concentrations of inflammatory mediators, lower plasma NGAL and reversal of sepsis-associated depletion of intra-renal T cell and myeloid cell subpopulations. Hierarchical clustering analysis of all 48-h serum/plasma analytes demonstrated partial co-clustering of repeated-dose hUC-MSC CLP animals with a Sham group but did not reveal a distinct signature of response to therapy. It was concluded that repeated doses of CD362-selected hUC-MSCs are required to modulate systemic and local immune/inflammatory events in polymicrobial sepsis and SA-AKI. Inter-individual variability and lack of effect of single dose MSC administration in the CLP model are consistent with observations to date from early-phase clinical trials.

An update on host immunity correlates and prospects of re-infection in COVID-19.

Reinfection with SARS-CoV-2 is not frequent yet the incidence rate of it is increasing globally owing to the slow emergence of drift variants that pose a perpetual threat to vaccination strategies and have a greater propensity for disease reoccurrence. Long-term protection against SARS-CoV-2 reinfection relies on the induction of the innate as well as the adaptive immune response endowed with immune memory. However, a multitude of factors including the selection pressure, the waning immunity against SARS-CoV-2 over the first year after infection possibly favors evolution of more infectious immune escape variants, amplifying the risk of reinfection. Additionally, the correlates of immune protection, the novel SARS-CoV-2 variants of concern (VOC), the durability of the adaptive and mucosal immunity remain major challenges for the development of therapeutic and prophylactic interventions. Interestingly, a recent body of evidence indicated that the gastrointestinal (GI) tract is another important target organ for SARS-CoV-2 besides the respiratory system, potentially increasing the likelihood of reinfection by impacting the microbiome and the immune response via the gut-lung axis. In this review, we summarized the latest development in SARS-CoV-2 reinfection, and explored the untapped potential of trained immunity. We also highlighted the immune memory kinetics of the humoral and cell-mediated immune response, genetic drift of the emerging viral variants, and discussed the current challenges in vaccine development. Understanding the dynamics and the quality of immune response by unlocking the power of the innate, humoral and cell-mediated immunity during SARS-CoV-2 reinfection would open newer avenues for drug discovery and vaccine designs.

Effects of mesenchymal stromal cells on regulatory T cells: Current understanding and clinical relevance.

The immunomodulatory potential of mesenchymal stromal cells (MSCs) and regulatory T cells (T-reg) is well recognized by translational scientists in the field of regenerative medicine and cellular therapies. A wide range of preclinical studies as well as a limited number of human clinical trials of MSC therapies have not only shown promising safety and efficacy profiles but have also revealed changes in T-reg frequency and function. However, the mechanisms underlying this potentially important observation are not well understood and, consequently, the optimal strategies for harnessing MSC/T-reg cross-talk remain elusive. Cell-to-cell contact, production of soluble factors, reprogramming of antigen presenting cells to tolerogenic phenotypes, and induction of extracellular vesicles ("exosomes") have emerged as possible mechanisms by which MSCs produce an immune-modulatory milieu for T-reg expansion. Additionally, these two cell types have the potential to complement each other's immunoregulatory functions, and a combinatorial approach may exert synergistic effects for the treatment of immunological diseases. In this review, we critically assess recent translational research related to the outcomes and mechanistic basis of MSC effects on T-reg and provide a perspective on the potential for this knowledge base to be further exploited for the treatment of autoimmune disorders and transplants.

Decoding intrathecal immunoglobulins and B cells in the CNS: their synthesis, function, and regulation.

The discovery of an active lymphatic system in the meninges (dura mater) has opened up a wide range of possibilities for the role of CNS immunoglobulins in brain development in early fetal life or during infancy. The antibody-dependent and -independent functions of B cells in the immunopathogenesis of multiple sclerosis are not new to immunologists, yet their role in other neurodegenerative disorders such as Alzheimer's and Parkinson's disease is incompletely understood. Deep cervical lymph nodes have emerged as a candidate site for autosensitization against CNS antigens and have been shown to provide the right kind of milieu for the dynamic interaction of antigen-presenting cells, B cells, and T cells. The presence of different B cells in the lymph nodes and the production of natural autoantibodies by B-1 cells have definitely unlocked another piece of the puzzle. At a time when CD19 and CD20 monoclonal antibodies have shown remarkable results in ameliorating the relapse and progression of multiple sclerosis, it is imperative to dissect out the diversity in B cell populations inside the CNS to identify new targets to improve current treatment regimens for neurodegenerative diseases. This review highlights the origin, migration, function, and regulation of B cells and the production of intrathecal immunoglobulins considering the previous and current findings and taking into account the differences between a healthy state and the changes that occur during an inflammatory or autoimmune response.

Comparative Proliferation Capacity of Gag-C-Specific Naive and Memory CD4+ and CD8+ T Lymphocytes in Rapid, Viremic Slow, and Slow Progressors During Human Immunodeficiency Virus Infection.

The exact cause of altered dynamics in T cells compartment during HIV infection remains elusive to date. In this longitudinal study, the proliferation frequency of different T cell subsets was investigated in untreated HIV-1-infected Indian individuals stratified as rapid (R), viremic slow (VS), slow (S) progressors, and healthy controls. Ten healthy and 20 treatment-naive HIV-1-infected individuals were enrolled. Expression of Ki67 nuclear antigen was examined on HIV-specific T cell subsets in peripheral blood lymphocytes. Upon stimulation with HIV-1 Gag-C peptide pools, effector memory (EM) CD4 T cells (R vs. S, EM CD4, p < 0.05) of R progressors proliferated significantly compared with those of S progressors at baseline. However, central memory (CM) CD8 T cell subsets proliferated significantly in VS and S progressors compared with those in R progressors, wherein highest proliferation frequency of EM CD8 T cells was observed. At follow-up visit, the proliferation frequency of naive CD8 T cells was significantly higher in R progressors than S progressors (R vs. S naive CD8, p < 0.05). The findings suggest altered dynamics of different CD4+ and CD8+ T cell subsets in R, VS, and S progressors. The increase in CM T cell proliferation in VS and S progressors could be attributed to slower progression of the HIV infection. Hence, treatment strategies must be focused on restoring the homeostatic balance to restore T cell functionality.

ETS1 and PAX5 transcription factors recruit AID to Igh DNA.

B lymphocytes optimize antibody responses by class switch recombination (CSR), which changes the expressed constant region exon of the immunoglobulin heavy chain (IgH), and by somatic hypermutation (SH) that introduces point mutations in the variable regions of the antibody genes. Activation-induced cytidine deaminase (AID) is the key mutagenic enzyme that initiates both these antibody diversification processes by deaminating cytosine to uracil. Here we asked the question if transcription factors can mediate the specific targeting of the antibody diversification by recruiting AID. We have recently reported that AID is together with the transcription factors E2A, PAX5 and IRF4 in a complex on key sequences of the Igh locus. Here we report that also ETS1 is together with AID in this complex on key sequences of the Igh locus in splenic B cells of mice. Furthermore, we show that both ETS1 and PAX5 can directly recruit AID to DNA sequences from the Igh locus with the specific binding site for the transcription factor. Taken together, our findings support the notion of a targeting mechanism for the selective diversification of antibody genes with limited genome wide mutagenesis by recruitment of AID by PAX5 and ETS1 in a transcription factor complex.

Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination.

B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces point mutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also that BCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH- and CSR-coupled proteins on switch regions of the Igh locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

Impact of HIV infection and highly active antiretroviral therapy (HAART) on B cell subpopulations in children.

B-cells play an important role in defending children against various infections. In view of scare data, we undertook this prospective cohort study to describe B cell compartment in HIV infected children (<5 years of age) and the effect of HAART on B cell subpopulations. HIV infected children (<5 years) from Pediatric HIV services of the Department of Pediatrics, All India Institute of Medical Sciences, New Delhi, were recruited (April 2012-December 2015). The enrolled HIV-1 infected children (n = 59) were followed up regularly for 12 months; healthy controls (n = 51) included HIV uninfected children with no major illness. Flow cytometry was performed on fresh EDTA-treated blood samples to characterize B cell subpopulations. In HIV-infected children, marked depletion of naive (P = 0.003), non-switched memory (P = 0.02), mature (P = 0.0005), resting memory (P < 0.0001) B cells, and expansion of double negative memory (P < 0.0001), activated memory (P < 0.0001) and tissue like memory (P < 0.0001) B cells were observed as compared to healthy controls. In children started on HAART, at the end of 12 months of therapy, frequencies of non-switched memory (P = 0.04), switched memory (P = 0.01), and resting memory (P = 0.003) B cells were lower; activated memory (P = 0.04), and tissue-like memory (P = 0.0001) B cells were still higher than healthy controls. HIV infection resulted in reduced memory B cells in HIV infected children. Following HAART, there was normalization of some B cell subpopulations. The study emphasizes the need of re-vaccination in HIV infected children to maintain the memory B cell pool and adequate humoral immune response against infections.

CNS: Not an immunoprivilaged site anymore but a virtual secondary lymphoid organ.

The cardinal dogma of central nervous system (CNS) immunology believed brain is an immune privileged site, but scientific evidences gathered so far have overturned this notion proving that CNS is no longer an immune privileged site, but rather an actively regulated site of immune surveillance. Landmark discovery of lymphatic system surrounding the duramater of the brain, made possible by high resolution live imaging technology has given new dimension to neuro-immunology. Here, we discuss the immune privilege status of CNS in light of the previous and current findings, taking into account the differences between a healthy state and changes that occur during an inflammatory response. Cerebrospinal fluid (CSF) along with interstitial fluid (ISF) drain activated T cells, natural killer cells, macrophages and dendritic cells from brain to regional lymph nodes present in the head and neck region. To keep an eye on inflammation, this system hosts an army of regulatory T cells (CD25+ FoxP3+) that regulate T cell hyper activation, proliferation and cytokine production. This review is an attempt to fill the gaps in our understanding of neuroimmune interactions, role of innate and adaptive immune system in maintaining homeostasis, interplay of different immune cells, immune tolerance, knowledge of communication pathways between the CNS and the peripheral immune system and lastly how interruption of immune surveillance leads to neurodegenerative diseases. We envisage that discoveries should be made not only to decipher underlying cellular and molecular mechanisms of immune trafficking, but should aid in identifying targeted cell populations for therapeutic intervention in neurodegenerative and autoimmune disorders.

Comparative evaluation of microbial translocation products (LPS, sCD14, IgM Endocab) in HIV-1 infected Indian individuals.

BACKGROUND: Microbial translocation of lipopolysaccharides (LPS), soluble CD14 (sCD14) and IgM Endocab levels have been reported to be associated with disease progression in HIV-1 infection. In this longitudinal study, plasma levels of different microbially translocated products (LPS, sCD14, Endocab) was investigated in HIV-1 infected Indian Individuals stratified as Rapid (R), Viremic slow (VS), Slow progressors (S) and healthy controls. METHOD: Ten healthy and twenty HIV-1 infected individuals were enrolled. Plasma levels of LPS, sCD14, Endocab was examined using commercially available Limulus Amebocyte assay and enzyme-linked immunosorbent assay (ELISA) enzyme linked immunosorbant assay. RESULTS: Elevated levels of sCD14, IgM EndoCab and LPS were observed during HIV-1 infection compared to healthy controls. Rapid progressors had higher levels of sCD14, IgM EndoCab, LPS (median% 1553, 3596, 202.2) compared to viremic slow, slow progressors and healthy controls both at baseline and follow up visits. At baseline, LPS correlated positively with IgM Endocab and negatively with sCD14 levels while at follow-up, significant positive correlation was observed between IgM Endocab and sCD14 (IgM EndoCab r = 0.490, p = 0.05; sCD14 r = 0.051, p = 0.830). Plasma levels of sCD14 correlated positively with viral load in rapid, viremic slow and slow progressors while CD + T cell count correlated positively with sCD14 and IgM EndoCab levels in viremic slow and slow progressors. CONCLUSION: Our findings indicate that elevated levels of sCD14, IgM EndoCab and LPS in HIV-1 infected individuals are strong predictors of disease progression and could be considered as candidate biomarkers for disease monitoring.

Cross-Reactive Potential of HIV-1 Subtype C-Infected Indian Individuals Against Multiple HIV-1 Potential T Cell Epitope Gag Variants.

Vaccine immunogen with expanded T cell coverage for protection against HIV-1 diversity is the need of the hour. This study was undertaken to examine the ability of T cells to respond to a broad spectrum of potential T cell epitope (PTE) peptides containing variable as well as conserved sequences that would most accurately reflect immune responses to different circulating strains. Set of 320 PTE peptides were pooled in a matrix format that included 40 pools of 32 peptides per pool. These pools were used in interferon-γ enzyme-linked immunospot assay for screening and confirmation of HIV-1 PTE Gag-specific T cell immune responses in 34 HIV-1 seropositive Indian individuals. "Deconvolute This" software was used for result analysis. The dominant target in terms of magnitude and breadth of responses was observed to be the p24 subunit of Gag protein. Of the 34 study subjects, 26 (77%) showed a response to p24 PTE Gag peptides, 17 (50%) to p17, and 17 (50%) responded to p15 PTE peptides. The total breadth and magnitude of immune response ranged from 0.75 to 14.50 and 95.02 to 1,103 spot-forming cells/106 cells, respectively. Seventy-six peptides located in p24 Gag were targeted by 77% of the study subjects followed by 51 peptides in p17 Gag and 46 peptides in p15 Gag with multiple variants being recognized. Maximum study participants recognized PTE peptide sequence Gag271→285NKIVRMYSPVSILDI located in p24 Gag subunit. T cells from HIV-1-infected individuals can recognize multiple PTE peptide variants, although the magnitude of the responses can vary greatly across these variants.

Comparative evaluation of a reverse transcriptase based assay for HIV-1 viral load quantitation in resource limited settings.

Molecular viral load assays are routinely used in high income countries for monitoring the copy number of human immunodeficiency virus (HIV) RNA. However, they require sophisticated facilities and expensive reagents and instruments. Hence, their routine use for patients belonging to resource limited settings is difficult and a low cost alternative is the need of the hour. This was a cross sectional study that analyzed and compared a reverse transcriptase enzyme based assay (Cavidi ExaVir Load version 3) with a real time polymerase chain reaction (PCR) assay (Roche COBAS TaqMan) in resource limited settings with subtype C predominance. The study included 75 HIV-1 positive treatment naïve patients whose CD4+ T lymphocytes count was estimated using BD FACS system and viral loads were quantified using both Cavidi ExaVir Load assay version 3 and Roche COBAS TaqMan Real Time PCR assay. The statistical analysis was performed using the Graph Pad Prism 5 software. The difference in the mean log10 viral load values was found to be 0.2log10copies/ml. The Bland Altman plot showed a clustering of viral load values toward the lower copy range. 78% of the samples had an agreement of ≤0.5 log10 copies/ml and 90.74% of the samples had an agreement of ≤1 log10 copies/ml. Both the assays showed a trend of negative correlation with the CD4+ T cell counts. The study found that ExaVir Load assay can be used as an alternative to the existing molecular assays in resource limited settings for the purpose of routine viral load measurement and monitoring treatment response.

The enduring tale of T cells in HIV immunopathogenesis.

HIV continues to be a major health problem worldwide even today. Owing to the intricate nature of its interactions with the immune system, HIV has remained an enigma that cleverly utilizes the host machinery to survive. Its ability to evade the host immune system, at both levels, innate and adaptive, allows the pathogen to replicate and transmit from one host to another. It has been shown that HIV has multipronged effects especially on the adaptive immunity, with CD4+ T cells being the worst affected T cell populations. Various analyses have revealed that the exposure to HIV results in clonal expansion and excessive activation of the immune system. Also, an abnormal process of differentiation has been observed suggestive of an alteration and blocks in the maturation of various T cell subsets. Additionally, HIV has shown to accelerate immunosenescence and exhaustion of the overtly activated T cells. Apart from causing phenotypic changes, HIV has adverse effects on the functional aspect of the immune system, with evidences implicating it in the loss of the capacity of T cells to secrete various antiviral cytokines and chemokines. However, there continues to be many aspects of the immunopathogenesis of HIV that are still unknown and thus require further research to convert the malaise of HIV into a manageable epidemic.