Inhibition of caspase pathways limits CD4+ T cell loss and restores host anti-retroviral function in HIV-1 infected humanized mice with augmented lymphoid tissue.

The study of HIV infection and pathogenicity in physical reservoirs requires a biologically relevant model. The human immune system (HIS) mouse is an established model of HIV infection, but defects in immune tissue reconstitution remain a challenge for examining pathology in tissues. We utilized exogenous injection of the human recombinant FMS-like tyrosine kinase 3 ligand (rFLT-3 L) into the hematopoietic stem cell (HSC) cord blood HIS mouse model to significantly expand the total area of lymph node (LN) and the number of circulating human T cells. The results enabled visualization and quantification of HIV infectivity, CD4 T cell depletion and other measures of pathogenesis in the secondary lymphoid tissues of the spleen and LN. Treatment with the Caspase-1/4 inhibitor VX-765 limited CD4+ T cell loss in the spleen and reduced viral load in both the spleen and axillary LN. In situ hybridization further demonstrated a decrease in viral RNA in both the spleen and LN. Transcriptomic analysis revealed that in vivo inhibition of caspase-1/4 led to an upregulation in host HIV restriction factors including SAMHD1 and APOBEC3A. These findings highlight the use of rFLT-3 L to augment human immune system characteristics in HIS mice to support investigations of HIV pathogenesis and test host directed therapies, though further refinements are needed to further augment LN architecture and cellular populations. The results further provide in vivo evidence of the potential to target inflammasome pathways as an avenue of host-directed therapy to limit immune dysfunction and virus replication in tissue compartments of HIV+ persons.

Combination of Cystatins 9 and C Modulates Serum Biomarkers Associated with Inflammation and Provides Prophylactic as Well as Long-Term Protection against Multidrug-Resistant Klebsiella pneumoniae.

We have identified recombinant human cystatins 9 (rCST9) and C (rCSTC) as a combination immunotherapeutic treatment against multidrug-resistant (MDR) New Delhi metallo-ß-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae We evaluated the lasting protection of rCST9/rCSTC treatment against MDR NDM-1 K. pneumoniae pneumonia. Results showed that rCST9/rCSTC treatment modulated endogenous serum biomarkers, cystatins 9 and C and amyloid A, associated with poor patient outcomes and provided prophylactic and long-term protection in a murine model of pneumonia.

Cystatins 9 and C as a Novel Immunotherapy Treatment That Protects against Multidrug-Resistant New Delhi Metallo-Beta-Lactamase-1-Producing Klebsiella pneumoniae.

Multidrug-resistant (MDR) bacterial pneumonia can induce dysregulated pulmonary and systemic inflammation leading to morbidity and mortality. Antibiotics to treat MDR pathogens do not function to modulate the extent and intensity of inflammation and can have serious side effects. Here we evaluate the efficacy of two human cysteine proteinase inhibitors, cystatin 9 (CST9) and cystatin C (CSTC), as a novel immunotherapeutic treatment to combat MDR New Delhi metallo-beta-lactamase-1 (NDM-1)-producing Klebsiella pneumoniae Our results showed that mice infected intranasally (i.n.) with a 90% lethal dose (LD90) challenge of NDM-1 K. pneumoniae and then treated with the combination of human recombinant CST9 (rCST9) and rCSTC (rCSTs; 50 pg of each i.n. at 1 h postinfection [p.i.] and/or 500 pg of each intraperitoneally [i.p.] at 3 days p.i.) had significantly improved survival compared to that of infected mice alone or infected mice treated with individual rCSTs (P < 0.05). Results showed that both of our optimal rCST treatment regimens modulated pulmonary and systemic proinflammatory cytokine secretion in the serum, lungs, liver, and spleen in infected mice (P < 0.05). Treatment also significantly decreased the bacterial burden (P < 0.05) while preserving lung integrity, with reduced inflammatory cell accumulation compared to that in infected mice. Further, rCST treatment regimens reduced lipid peroxidation and cell apoptosis in the lungs of infected mice. Additionally, in vitro studies showed that rCSTs (50 or 500 pg of each) directly decreased the viability of NDM-1 K. pneumoniae In conclusion, the data showed that rCST9/rCSTC worked synergistically to modulate host inflammation against MDR NDM-1 K. pneumoniae pneumonia, which significantly improved survival. Therefore, rCST9/rCSTC is a promising therapeutic candidate for the treatment of bacterial pneumonia.

Therapeutic Modulation of Arginase with nor-NOHA Alters Immune Responses in Experimental Mouse Models of Pulmonary Tuberculosis including in the Setting of Human Immunodeficiency Virus (HIV) Co-Infection.

L-arginine metabolism is strongly linked with immunity to mycobacteria, primarily through the antimicrobial activity of nitric oxide (NO). The potential to modulate tuberculosis (TB) outcomes through interventions that target L-arginine pathways are limited by an incomplete understanding of mechanisms and inadequate in vivo modeling. These gaps in knowledge are compounded for HIV and Mtb co-infections, where activation of arginase-1 due to HIV infection may promote survival and replication of both Mtb and HIV. We utilized in vitro and in vivo systems to determine how arginase inhibition using Nω-hydroxy-nor-L-arginine (nor-NOHA) alters L-arginine pathway metabolism relative to immune responses and disease outcomes following Mtb infection. Treatment with nor-NOHA polarized murine macrophages (RAW 264.7) towards M1 phenotype, increased NO, and reduced Mtb in RAW macrophages. In Balb/c mice, nor-NOHA reduced pulmonary arginase and increased the antimicrobial metabolite spermine in association with a trend towards reduced Mtb CFU in lung. In humanized immune system (HIS) mice, HIV infection increased plasma arginase and heightened the pulmonary arginase response to Mtb. Treatment with nor-NOHA increased cytokine responses to Mtb and Mtb/HIV in lung tissue but did not significantly alter bacterial burden or viral load. Our results suggest that L-arginine pathway modulators may have potential as host-directed therapies to augment antibiotics in TB chemotherapy.