Ashok Kumar

The eye is highly susceptible to inflammation-induced tissue damage; however, the mechanisms that drive inflammation resolution during ocular infections remain unclear. In this study, we utilize a murine model of intraocular bacterial infection (S. aureus-induced endophthalmitis) and lipidomics analysis to uncover a critical role of pro-resolving lipid mediators, particularly resolvin D1 (RvD1), in resolving inflammation and restoring ocular tissue homeostasis and vision. RvD1 protects mouse eyes from severe endophthalmitis by enhancing bacterial clearance, suppressing intraocular inflammation, and preserving retinal structure and function. Pharmacological inhibition of formyl peptide receptor 2 (FPR2) reveals that RvD1’s protective effects mainly rely on FPR2 signaling. Unexpectedly, RvD1 is unable to resolve inflammation or protect the eye in the absence of Toll-like receptor 2 (TLR2), a critical pattern recognition receptor in ocular S. aureus infections. These findings reveal an unrecognized interplay between TLR2 and FPR2 signaling, including their mutual regulation and physical receptor interactions during bacterial infection. Overall, these findings provide new insights into the coordinated roles of TLR2 and FPR2 in resolving inflammation and protecting the eye during bacterial infections.

The rising incidence of drug-resistant ocular pathogens highlights the critical need for innovative therapeutic strategies. Here, we developed an antibacterial nanoformulation by combining silver with an antibiotic (vancomycin) and evaluated its efficacy using in vitro, ex vivo, and in vivo models of ocular Staphylococcus aureus infections. Silver nanoparticles were synthesized through chemical reduction using silver nitrate and sodium citrate, subsequently loaded with vancomycin, and coated with chitosan to impart a positive surface charge. These nanoformulations were tested for their antimicrobial activity against S. aureus, including methicillin-resistant S. aureus (MRSA) and clinical isolates. The toxicity was evaluated in cultured human retinal Müller glia cells and mouse eyes. Ex vivo studies were performed using porcine/goat eyes and human vitreous. Therapeutic efficacy was evaluated in a mouse model of S. aureus endophthalmitis. Three formulations (AgNPs, CAgNPs, and CAgVNPs) ranged in size from 60 to 70 nm and demonstrated efficient vancomycin loading with controlled release in acidic conditions. Antimicrobial testing revealed synergistic antibacterial effects of nanoformulations against S. aureus, including MRSA and clinical isolates, resulting in a two- to fourfold reduction in minimum inhibitory concentration compared to vancomycin. Both in vitro and in vivo assays showed negligible cytotoxicity. All nanoformulations ameliorated the severity of S. aureus endophthalmitis, with CAgVNPs showing a marked reduction in bacterial burden (two- to sevenfold) and inflammatory cytokines (three- to fivefold). Moreover, CAgVNP-treated eyes exhibited reduced cell death and retinal tissue damage. Our study demonstrates that the synergistic activity of chitosan-coated silver nanoparticles and antibiotics represents a promising therapeutic strategy for treating resistant ocular bacterial infections.

Bacterial and fungal pathogens are major causes of infectious endophthalmitis following eye surgery or trauma, often leading to vision impairment or blindness. The distinct clinical outcomes observed in bacterial and fungal endophthalmitis suggest differences in host immune responses. To investigate these differences, we utilized cytokine arrays and murine models of bacterial ( Staphylococcus aureus ) and fungal ( Aspergillus fumigatus ) endophthalmitis. Our analysis revealed that cytokine responses peaked in bacterial infections at 12–24 h, whereas fungal infections exhibited a delayed peak at 48 h. Several inflammatory mediators, including MMP9, MMP3, CD14, LIX, LCN2, retinol-binding protein 4, ICAM1, and VCAM1, were differentially elevated. Notably, interleukin-33 (IL-33) levels peaked early in bacterial infections but continued to rise throughout all time points in fungal endophthalmitis. Analysis of patient vitreous samples further confirmed higher levels of IL-33 in bacterial ( n =40) and fungal ( n =20) endophthalmitis cases. Functional studies in IL-33-deficient mice revealed an increased fungal burden and elevated TNF-α and IL-6 levels, but bacterial endophthalmitis severity remains largely unaffected. Additionally, bone marrow-derived macrophages from IL-33 ^−/− mice exhibited increased cell death in response to fungal and bacterial infection. Our findings reveal divergent innate immune responses between bacterial and fungal endophthalmitis and emphasize the immunomodulatory function of IL-33 in ocular infections.

Mpox virus (MPXV), a re-emerging zoonotic threat, has caused outbreaks in non-endemic regions through respiratory, sexual, and close-contact transmission. The increased transmissibility of Clade IIb fueled the 2022 global outbreak, with 2024 Clade Ib spread in the Democratic Republic of Congo further escalating concern. Both outbreaks were declared public health emergencies by the WHO. Although tecovirimat (TPOXX) has been used off-label for Mpox, its limited effectiveness highlights the critical need for newer antivirals for MPXV. We conducted high-throughput antiviral drug screening using a host-directed kinase inhibitor library composed of 2,750 compounds against 2022 Clade IIb MPXV. Our primary screen identified 138 compounds preventing MPXV cytopathic effects, including multiple inhibitors of EGFR, PI3K-mTOR, and Ras/Raf, as well as apoptosis and autophagy regulators. Secondary and tertiary screenings yielded a shortlist of potent, nontoxic antiviral compounds that inhibited MPXV replication. Three selected compounds, IRAK4-IN-6, SM-7368, and KRAS inhibitor-10, reduced MPXV-induced cell death in primary human epidermal keratinocytes. IRAK4-IN-6 and SM-7368 were also found to modulate NF-κB and STING signaling. Furthermore, these compounds were found effective in reducing skin lesions and viral burden in a mouse model of MPXV skin infection. Together, our study reveals new classes of antiviral compounds against MPXV, offering promising candidates for future clinical development.

Mpox virus (MPXV), a re-emerging zoonotic threat, has caused outbreaks in non-endemic regions through respiratory, sexual, and close-contact transmission. The increased transmissibility of Clade IIb fueled the 2022 global outbreak, with 2024 Clade Ib spread in the Democratic Republic of Congo further escalating concern. Both outbreaks were declared public health emergencies by the WHO. Although tecovirimat (TPOXX) has been used off-label for Mpox, its limited effectiveness highlights the critical need for newer antivirals for MPXV. We conducted high- throughput antiviral drug screening using a host-directed kinase inhibitor library composed of 2,750 compounds against 2022 Clade IIb MPXV. Our primary screen identified 138 compounds preventing MPXV cytopathic effects, including multiple inhibitors of EGFR, PI3K-mTOR, and Ras/Raf, as well as apoptosis and autophagy regulators. Secondary and tertiary screenings yielded a shortlist of potent, nontoxic antiviral compounds that inhibited MPXV replication. Three selected compounds, IRAK4-IN-6, SM-7368, and KRAS inhibitor-10, reduced MPXV-induced cell death in primary human epidermal keratinocytes. IRAK4-IN-6 and SM-7368 were also found to modulate NF-κB and STING signaling. Furthermore, these compounds were found effective in reducing skin lesions and viral burden in a mouse model of MPXV skin infection. Together, our study reveals new classes of antiviral compounds against MPXV, offering promising candidates for future clinical development.

Metabolomic profiling of aqueous humor from primary open-angle glaucoma (POAG) patients using targeted metabolomics analysis and assessment of the potential anti-neuroinflammatory and neuroprotective roles of key dysregulated metabolites in a mouse model of retinal neuroinflammation. A targeted metabolomics was performed on aqueous humor from POAG patients (n = 19) and healthy subjects (n = 10) via LC-MS/MS. In vitro neuroprotection studies were performed using a mouse cone photoreceptor cell line (661W) exposed to oxidative stress. For in vivo therapeutic studies, a few key dysregulated metabolites were delivered either topically via extracellular vesicle (EV)-mediated delivery or intravitreally into a C57BL/6 mouse model of retinal neuroinflammation. The neuroprotective and anti-neuroinflammatory properties were determined in the presence and absence of metabolites through pattern electroretinography, TUNEL, and quantitative PCR analyses. Among the 135 endogenous metabolites identified, 31 metabolites showed significant dysregulation in POAG. Metabolite set enrichment analysis revealed that these altered metabolites were associated with dysregulation of multiple key cellular pathways, including glycolysis, pentose phosphate pathway, short-/long-chain fatty acid metabolism, mitochondrial β-oxidation, and electron transport chain under glaucomatous conditions. Among these differentially expressed metabolites, a putative neuromodulator (agmatine) and a vitamin (thiamine) significantly decreased in POAG patients. Intravitreal or EV-mediated topical delivery of agmatine and thiamine significantly reduced the inflammatory response and protected retinal ganglion cell function against neuroinflammatory damage in the mouse retina. Agmatine and thiamine treatment also significantly protected photoreceptor cells from oxidative stress-induced cell death and attenuated the inflammatory cytokine response. Our results revealed significant metabolic alterations in POAG that affect key cellular functions. Agmatine and thiamine could be potential immunomodulatory or neuroprotective drugs to treat or prevent neuroinflammatory damage to the retina during glaucoma.