Although BCG vaccine protects infants from tuberculosis (TB), it has limited efficacy in adults against pulmonary TB. Further, HIV coinfection significantly increases the risk of developing active ...TB. In the lack of defined correlates of protection in TB disease, it is essential to explore immune responses beyond conventional CD4 T cells to gain a better understanding of the mechanisms of TB immunity.
Here, we evaluated unconventional lipid-reactive T cell responses in cynomolgus macaques following aerosol BCG inoculation and examined the impact of subsequent SIV infection on these responses. Immune responses to cellular lipids of
and
were examined ex vivo in peripheral blood and bronchioalveolar lavage (BAL).
Prior to BCG inoculation, innate-like IFN-γ responses to mycobacterial lipids were observed in T cells. Aerosol BCG exposure induced an early increase in frequencies of BAL γδT cells, a dominant subset of lipid-reactive T cells, along with enhanced IL-7R and CXCR3 expression. Further, BCG exposure stimulated greater IFN-γ responses to mycobacterial lipids in peripheral blood and BAL, suggesting the induction of systemic and local Th1-type response in lipid-reactive T cells. Subsequent SIV infection resulted in a significant loss of IL-7R expression on blood and BAL γδT cells. Additionally, IFN-γ responses of mycobacterial lipid-reactive T cells in BAL fluid were significantly lower in SIV-infected macaques, while perforin production was maintained through chronic SIV infection.
Overall, these data suggest that despite SIV-induced decline in IL-7R expression and IFN-γ production by mycobacterial lipid-reactive T cells, their cytolytic potential is maintained. A deeper understanding of anti-mycobacterial lipid-reactive T cell functions may inform novel approaches to enhance TB control in individuals with or without HIV infection.
HIV-associated inflammation has been implicated in the premature aging and increased risk of age-associated comorbidities in cART-treated individuals. However, the immune mechanisms underlying the ...chronic inflammatory state of cART-suppressed HIV infection remain unclear. Here, we investigated the role of γδT cells, a group of innate IL-17 producing T lymphocytes, in the development of systemic inflammation and leaky gut phenotype during cART-suppressed SIV infection of macaques. Plasma levels of inflammatory mediators, intestinal epithelial barrier disruption (IEBD) and microbial translocation (MT) biomarkers, and Th1/Th17-type cytokine functions were longitudinally assessed in blood and gut mucosa of SIV-infected, cART-suppressed macaques. Among the various gut mucosal IL-17/IL-22-producing T lymphocyte subsets including Th17, γδT, CD161
CD8
T, and MAIT cells, a specific decline in the Vδ2 subset of γδT cells and impaired IL-17/IL-22 production in γδT cells significantly correlated with the subsequent increase in plasma IEBD/MT markers (IFABP, LPS-binding protein, and sCD14) and pro-inflammatory cytokines (IL-6, IL-1β, IP10, etc.) despite continued viral suppression during long-term cART. Further, the plasma inflammatory cytokine signature during long-term cART was distinct from acute SIV infection and resembled the inflammatory cytokine profile of uninfected aging (inflammaging) macaques. Overall, our data suggest that during cART-suppressed chronic SIV infection, dysregulation of IL-17/IL-22 cytokine effector functions and decline of Vδ2 γδT cell subsets may contribute to gut epithelial barrier disruption and development of a distinct plasma inflammatory signature characteristic of inflammaging. Our results advance the current understanding of the impact of chronic HIV/SIV infection on γδT cell functions and demonstrate that in the setting of long-term cART, the loss of epithelial barrier-protective functions of Vδ2 T cells and ensuing IEBD/MT occurs before the hallmark expansion of Vδ1 subsets and skewed Vδ2/Vδ1 ratio. Thus, our work suggests that novel therapeutic approaches toward restoring IL-17/IL-22 cytokine functions of intestinal Vδ2 T cells may be beneficial in preserving gut epithelial barrier function and reducing chronic inflammation in HIV-infected individuals.
Gut dysbiosis is a common feature associated with the chronic inflammation of HIV infection. Toward understanding the interplay of chronic treated HIV infection, dysbiosis, and systemic inflammation, ...we investigated longitudinal fecal microbiome changes and plasma inflammatory markers in the nonhuman primate model. Following simian immunodeficiency virus (SIV) infection in rhesus macaques, significant changes were observed in several members of the phylum Firmicutes along with an increase in Bacteroidetes. Viral suppression with antiretroviral therapy (ART) resulted in an early but partial recovery of compositional changes and butyrate producing genes in the gut microbiome. Over the course of chronic SIV infection and long-term ART, however, the specific loss of
and
significantly correlated with an increase in plasma inflammatory cytokines including IL-6, G-CSF, I-TAC, and MIG. Further, the loss of
correlated with an increase in circulating biomarkers of gut epithelial barrier damage (IFABP) and microbial translocation (LBP and sCD14). As
and
are major short-chain fatty acid producing bacteria, their sustained loss during chronic SV-ART may contribute to gut inflammation and metabolic alterations despite effective long-term control of viremia. A better understanding of the correlations between the anti-inflammatory bacterial community and healthy gut barrier functions in the setting of long-term ART may have a major impact on the clinical management of inflammatory comorbidities in HIV-infected individuals.
Although most SARS-CoV-2 infections are mild, some patients develop systemic inflammation and progress to acute respiratory distress syndrome (ARDS). However, the cellular mechanisms underlying this ...spectrum of disease remain unclear. γδT cells are T lymphocyte subsets that have key roles in systemic and mucosal immune responses during infection and inflammation. Here we show that peripheral γδT cells are rapidly activated following aerosol or intra-tracheal/intra-nasal (IT/IN) SARS-CoV-2 infection in nonhuman primates. Our results demonstrate a rapid expansion of Vδ1 γδT cells at day1 that correlate significantly with lung viral loads during the first week of infection. Furthermore, increase in levels of CCR6 and Granzyme B expression in Vδ1 T cells during viral clearance imply a role in innate-like epithelial barrier-protective and cytotoxic functions. Importantly, the early activation and mobilization of circulating HLA-DR
CXCR3
γδT cells along with significant correlations of Vδ1 T cells with IL-1Ra and SCF levels in bronchoalveolar lavage suggest a novel role for Vδ1 T cells in regulating lung inflammation during aerosol SARS-CoV-2 infection. A deeper understanding of the immunoregulatory functions of MHC-unrestricted Vδ1 T cells in lungs during early SARS-CoV-2 infection is particularly important in the wake of emerging new variants with increased transmissibility and immune evasion potential.
Clinical measurements commonly used to evaluate overall health of laboratory animals including complete blood count, serum chemistry, weight, and immunophenotyping, differ with respect to age, ...development, and environment. This report provides comprehensive clinical and immunological reference ranges for pediatric rhesus macaques over the first year of life.
We collected and analyzed blood samples from 151 healthy rhesus macaques, aged 0-55 weeks, and compared mother-reared infants to two categories of nursery-reared infants; those on an active research protocol and those under derivation for the expanded specific-pathogen-free breeding colony. Hematology was performed on EDTA-anticoagulated blood using a Sysmex XT2000i, and serum clinical chemistry was performed using the Beckman AU480 chemistry analyzer. Immunophenotyping of whole blood was performed with immunofluorescence staining and subsequent flow cytometric analysis on a BD LSRFortessa. Plasma cytokine analysis was performed using a Millipore multiplex Luminex assay.
For hematological and chemistry measurements, pediatric reference ranges deviate largely from adults. Comparison of mother-reared and nursery-reared animals revealed that large differences depend on rearing conditions and diet. Significant differences found between two nursery-reared cohorts (research and colony animals) indicate large influences of experimental factors and anesthetic events on these parameters. Immune cells and cytokine responses presented with distinct patterns for infants depending on age, birth location, and rearing conditions.
Our results illustrate how the immune system changed over time and that there was variability among pediatric age groups. Reference ranges of results reported here will support interpretations for how infection and treatment may skew common immune correlates used for assessment of pathology or protection in research studies as well as help veterinarians in the clinical care of infant non-human primates. We highlighted the importance of using age-specific reference comparisons for pediatric studies and reiterated the utility of rhesus macaques as a model for human studies. Given the rapid transformation that occurs in multiple tissue compartments after birth and cumulative exposures to antigens as individuals grow, a better understanding of immunological development and how this relates to timing of infection or vaccination will support optimal experimental designs for developing vaccines and treatment interventions.
Abstract
Infection with SARS-CoV-2, the novel coronavirus responsible for the current COVID-19 pandemic, results in a wide range of pathologies from asymptomatic/mild outcome to fulminant pneumonia ...and progression to acute respiratory distress syndrome (ARDS). The cellular mechanisms underlying this spectrum of disease remain unknown. We investigated the dynamic changes in frequency and activation status of peripheral innate lymphocyte subsets in two different species of nonhuman primates. Cohorts of Rhesus macaques (n=8) and African Green monkeys (n=8) were infected with SARS-CoV-2 either by small particle or mucosal (IN/IT) route. Flow cytometric analysis of PBMCs revealed a rapid and significant increase in circulating γδ T cell frequencies and activation at d1 postexposure across all animals inoculated by aerosol, which was correlative with peak viral loads in lung lavage and Vδ2 T cell frequencies. Further, elevated GranzymeB expression in both Vδ1 and Vδ2 subsets of γδ T cells was suggestive of increased cytotoxic function in early infection. In contrast, mucosally-infected animals showed little change in circulating γδ T cell frequencies from d1 through d7 post-infection, nor were there remarkable differences in early MAIT and NK cell frequencies. Collectively, these data suggest γδ T cells may be early responders to deep lung SARS-CoV-2 infection and the kinetics and quality of immune response are dependent upon route of exposure despite resulting in similar peak viral loads from infection. Further understanding of γδ T cell functions within the pulmonary compartment during SARS-CoV-2-driven acute inflammation may reveal tractable therapeutic approaches to further mitigate ARDS-like responses in COVID-19 patients.
Abstract
Aging is characterized with a loss of hematopoietic tissue in bone marrow, systemic chronic inflammation, and higher susceptibility to infectious diseases. We previously showed the tightly ...regulated kinetics and massive daily production of neutrophils during homeostasis in rhesus macaques aged 3 to 19 years old. Here we extended that study to further investigate the effect of aging on kinetics and production of neutrophils in more elderly rhesus macaques. We observed a neutrophil kinetics shift and higher in-group variability in rhesus macaques above 20 years old (equivalent to 70 years or more in humans) by in vivo BrdU pulse-chase labeling. In a cross-sectional study, we also measured complete blood cell counts and plasma cytokine levels from a group of 126 outdoor-housed captive Indian-ancestry rhesus macaques between 2 to 23 years old and found that aging negatively correlated with neutrophil counts and positively correlated with plasma G-CSF levels. Hierarchical clustering analysis suggested that G-CSF was strongly associated with pro-inflammatory cytokines IL-1b and MIP-1a. In elderly rhesus macaques, neutrophils expressed less myeloperoxidase and there was a higher frequency of PMN-MDSCs compared to the young adult macaques. In summary, there appears to be a dysregulated feedback mechanism wherein increased levels of G-CSF failed to restore neutrophil production in elderly rhesus macaques that was associated with induced production of pro-inflammatory cytokines and earlier release of less mature neutrophils and PMN-MDSCs. Together, these findings may contribute to the understanding of the chronic inflammation and greater susceptibility to infectious diseases in the elderly.
Aging is characterized by a loss of bone marrow hematopoietic tissue, systemic chronic inflammation, and higher susceptibility to infectious and noninfectious diseases. We previously reported the ...tightly regulated kinetics and massive daily production of neutrophils during homeostasis in adult rhesus macaques aged 3 to 19 yr (equivalent to approximately 10 to 70 yr of age in humans). In the current study, we observed an earlier release of recently dividing neutrophils from bone marrow and greater in‐group variability of neutrophil kinetics based on in vivo BrdU labeling in a group of older rhesus macaques of 20–26 yr of age. Comparing neutrophil numbers and circulating cytokine levels in rhesus macaques spanning 2 to 26 yr of age, we found a negative correlation between age and blood neutrophil counts and a positive correlation between age and plasma G‐CSF levels. Hierarchic clustering analysis also identified strong associations between G‐CSF with the proinflammatory cytokines, IL‐1β and MIP‐1α. Furthermore, neutrophils from older macaques expressed less myeloperoxidase and comprised higher frequencies of polymorphonuclear myeloid‐derived suppressor cells (PMN‐MDSCs) compared to the young adult macaques. In summary, we observed an earlier release from bone marrow and a reduced production of neutrophils despite the increased levels of plasma G‐CSF, especially in the elderly rhesus macaques. This lower neutrophil production capacity associated with increased production of proinflammatory cytokines as well as an earlier release of less mature neutrophils and PMN‐MDSCs may contribute to the chronic inflammation and greater susceptibility to infectious and noninfectious diseases during aging.
Graphical
Dysregulated neutrophil kinetics, increasing G‐CSF levels, and higher frequency of PMN‐MDSCs accompany chronic inflammation during aging in rhesus macaques.
The development of chronic inflammation, called inflammaging, contributes to the pathogenesis of age-related diseases. Although it is known that both B and T lymphocyte compartments of the adaptive ...immune system deteriorate with advancing age, the impact of aging on immune functions of Th17-type CD161-expressing innate immune cells and their role in inflammaging remain incompletely understood. Here, utilizing the nonhuman primate model of rhesus macaques, we report that a dysregulated Th17-type effector function of CD161
+
immune cells is associated with leaky gut and inflammatory phenotype of aging. Higher plasma levels of inflammatory cytokines IL-6, TNF-α, IL-1β, GM-CSF, IL-12, and Eotaxin correlated with elevated markers of gut permeability including LPS-binding protein (LBP), intestinal fatty acid binding protein (I-FABP), and sCD14 in aging macaques. Further, older macaques displayed significantly lower frequencies of circulating Th17-type immune cells comprised of CD161
+
T cell subsets, NK cells, and innate lymphoid cells. Corresponding with the increased markers of gut permeability, production of the type-17 cytokines IL-17 and IL-22 was impaired in CD161
+
T cell subsets and NK cells, along with a skewing towards IFN-γ cytokine production. These findings suggest that reduced frequencies of CD161
+
immune cells along with a specific loss in Th17-type effector functions contribute to impaired gut barrier integrity and systemic inflammation in aging macaques. Modulating type-17 immune cell functions via cytokine therapy or dietary interventions towards reducing chronic inflammation in inflammaging individuals may have the potential to prevent or delay age-related chronic diseases and improve immune responses in the elderly population.
Dextrans have been used extensively as medical therapies and labeling agents in biomedical research to investigate the blood-brain barrier and CSF flow and absorption. Adverse effects from dextrans ...include anaphylactic reaction and dilation of the cerebral ventricles due to administration
into the subarachnoid space. This retrospective study describes 51 rhesus macaques (Macaca mulatta) that received dextran intrathecally. The purpose of intrathecal administration was to enable detection of long-lived, dextran-labeled macrophages and to study monocyte-macrophage
turnover in the CNS of SIV- or SHIV- infected and uninfected animals by using immunofluorescence. Of the 51 dextran-treated macaques, 8 that received dextran diluted in saline developed hydrocephalus; 6 of these 8 animals exhibited neurologic signs. In contrast, none of the macaques that received
intrathecal dextran diluted in PBS developed hydrocephalus. These data suggest the use of saline diluent and the duration of dextran exposure as potential factors contributing to hydrocephalus after intrathecal dextran in rhesus macaques.
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