Distinct cell death pathways induced by granzymes collectively protect against intestinal Salmonella infection.

Intestinal intraepithelial T lymphocytes (IEL) constitutively express high amounts of the cytotoxic proteases Granzymes (Gzm) A and B and are therefore thought to protect the intestinal epithelium against infection by killing infected epithelial cells. However, the role of IEL granzymes in a protective immune response has yet to be demonstrated. We show that GzmA and GzmB are required to protect mice against oral, but not intravenous, infection with Salmonella enterica serovar Typhimurium, consistent with an intestine-specific role. IEL-intrinsic granzymes mediate the protective effects by controlling intracellular bacterial growth and aiding in cell-intrinsic pyroptotic cell death of epithelial cells. Surprisingly, we found that both granzymes play non-redundant roles. GzmB-/- mice carried significantly lower burdens of Salmonella, as predominant GzmA-mediated cell death effectively reduced bacterial translocation across the intestinal barrier. Conversely, in GzmA-/- mice, GzmB-driven apoptosis favored luminal Salmonella growth by providing nutrients, while still reducing translocation across the epithelial barrier. Together, the concerted actions of both GzmA and GzmB balance cell death mechanisms at the intestinal epithelium to provide optimal control that Salmonella cannot subvert.

Granzyme B in aging and age-related pathologies.

Aging is a major risk factor for pathologies that manifest later in life. Much attention is devoted towards elucidating how prolonged environmental exposures and inflammation promote biological (accelerated) tissue aging. Granzymes, a family of serine proteases, are increasingly recognized for their emerging roles in biological aging and disease. Widely recognized as intracellular mediators of cell death, granzymes, particularly granzyme B (GzmB), also accumulate in the extracellular milieu of tissues with age, contributing to chronic tissue injury, inflammation, and impaired healing. Consequently, this has prompted the field to reconsider how GzmB regulation, accumulation, and proteolysis impact health and disease with age. While GzmB is observed in numerous age-related conditions, the current review focuses on mechanistic studies where proof-of-concept has been forwarded.

Distinct granzyme k expression in immune cells: a single-cell rna-seq meta-analysis.

BACKGROUND: Granzymes are essential serine proteases in cytotoxic T cells and natural killer (NK) cells, with GZMK's expression being less understood. This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. OBJECTIVE: This study aims to uncover GZMK expression profiles across various immune cell types using single-cell RNA sequencing meta-analysis. METHODS: We conducted a meta-analysis using cellxgene, an interactive data exploration platform developed by the Chan Zuckerberg Initiative. We focused on mature T cells, NK cells, B cells, and NKT cells. We also checked transcription factor binding sites at the granzyme gene promoter regions using JASPAR. Comparative analysis was also done using mouse single-cell RNA sequencing data. RESULTS: GZMK was the most lowly expressed in NK cells and mature NKT cells in most tissues except for colon and lymph nodes. In mature T cells, GZMK is similarly or more highly expressed than other granzymes. HBCA data revealed weak expression of GZMK in NK cells but strong expression in effector memory CD8-positive, alpha-beta T cells. Combined data shows no significant difference in GZMK expression between cell types. Subtype analysis shows that GZMK expression was higher in CD16-negative, CD56-bright NK cells when compared to CD16-positive, CD56-dim NK cells. We also identified unique transcription factor binding sites for GZMK. While this pattern in mouse data with low Gzmk expression in NK cells and higher T cells was repeated. CONCLUSION: GZMK expression is distinctively regulated among immune cells and tissues, with unique promoter regions and transcription factor binding sites contributing to this differential expression. These insights into GZMK's role in immune function and regulation offer potential therapeutic targets.

Peripheral Blood CD8+ T-Lymphocyte Immune Response in Benign and Subpopulations of Breast Cancer Patients.

Peripheral blood CD8+ T lymphocytes play a crucial role in cell-mediated immunity and tumor-related immune responses in breast cancer. In this study, label-free quantification analysis and gene set enrichment analysis (GSEA) of CD8+ T lymphocytes in the peripheral blood of benign patients and patients with different breast cancer (BC) subtypes, i.e., luminal A, luminal B, and triple-negative breast cancer (TNBC), were performed using nano-UHPLC and Orbitrap mass spectrometry. Differential protein expression in CD8+ T lymphocytes revealed significant downregulation (log2 FC ≥ 0.38 or ≤-0.38, adj. p < 0.05), particularly in proteins involved in cytotoxicity, cytolysis, and proteolysis, such as granzymes (GZMs) and perforin 1 (PRF1). This downregulation was observed in the benign group (GZMH, GZMM, and PRF1) and luminal B (GZMA, GZMH) subtypes, whereas granzyme K (GZMK) was upregulated in TNBC in comparison to healthy controls. The RNA degradation pathway was significantly downregulated (p < 0.05, normalized enrichment score (NES) from -1.47 to -1.80) across all BC subtypes, suggesting a potential mechanism for regulating gene expression during T cell activation. Also, the Sm-like proteins (LSM2, LSM3, and LSM5) were significantly downregulated in the RNA degradation pathway. Proteomic analysis of CD8+ T lymphocytes in peripheral blood across different breast cancer subtypes provides a comprehensive view of the molecular mechanisms of the systemic immune response that can significantly contribute to advancements in the diagnosis, treatment, and prognosis of this disease.

Granzyme K mediates IL-23-dependent inflammation and keratinocyte proliferation in psoriasis.

Psoriasis is an inflammatory disease with systemic manifestations that most commonly presents as itchy, erythematous, scaly plaques on extensor surfaces. Activation of the IL-23/IL-17 pro-inflammatory signaling pathway is a hallmark of psoriasis and its inhibition is key to clinical management. Granzyme K (GzmK) is an immune cell-secreted serine protease elevated in inflammatory and proliferative skin conditions. In the present study, human psoriasis lesions exhibited elevated GzmK levels compared to non-lesional psoriasis and healthy control skin. In an established murine model of imiquimod (IMQ)-induced psoriasis, genetic loss of GzmK significantly reduced disease severity, as determined by delayed plaque formation, decreased erythema and desquamation, reduced epidermal thickness, and inflammatory infiltrate. Molecular characterization in vitro revealed that GzmK contributed to macrophage secretion of IL-23 as well as PAR-1-dependent keratinocyte proliferation. These findings demonstrate that GzmK enhances IL-23-driven inflammation as well as keratinocyte proliferation to exacerbate psoriasis severity.

Local Low-dose Anti-PD-L1 Antibodies Improve Antitumor Effects in Oral Squamous Cell Carcinoma.

BACKGROUND/AIM: Immune checkpoint inhibitors are highly effective for treating recurrent and metastatic head and neck cancers. However, they require systemic administration and are associated with immune-related adverse events (irAEs). Reducing therapeutic antibody doses to prevent irAEs is challenging. MATERIALS AND METHODS: Mouse buccal mucosa squamous cell carcinoma cells (Sq-1979) were transplanted into the backs of mice to induce tumors. The antitumor efficacy and tumor immunohistological environment in tumor-bearing mice were compared after administering a standard dose of programmed death-ligand 1 (PD-L1) antibodies systemically (200 mg/body) or 1/10th of the standard dose (20 mg/body) directly to tumors. Mice received four doses of antibody administered in 3-day intervals. Tumor reduction rates and antitumor efficacies were evaluated 21 days after initiating treatment. CD8+T cell counts and PD-L1, PD-1, perforin, and granzyme B levels; CD25 and Foxp3 expression levels; and tumor Tregs were assessed in the resected subcutaneous tumors. RESULTS: The antitumor efficacies in the local low-dose and systemic standard-dose groups were compared with that of the control group. The efficacies of the two treatment groups were similar, and both treatment groups revealed significant antitumor effects compared to the control group. Perforin and granzyme B levels were higher in the local low-dose group (p<0.05). CONCLUSION: Local low-dose administration of anti-PD-L1 antibodies exhibits antitumor efficacy similar to systemic standard-dose administration suggesting that local low-dose administration is useful for treating oral squamous cell carcinoma.

Detection of Granzyme B-associated Binding Targets in Peripheral Blood Samples of Hosts in Sickness and in Health Using a Granzyme B-like Peptide Fluorescent Conjugate (GP1R).

Granzyme B, mostly expressed by cytotoxic T lymphocytes in the fight against cancer and infection, is known to induce cell death based on its active enzymatic activity as a serine protease. Recent studies showed cytotoxicity of a non-enzymatic granzyme B-like peptide (also referred to as granzyme B-associated peptide or GP1 in this report) in tumor cells and presence of binding targets for GP1R (i.e., GP1 conjugated with rhodamine fluorochrome) in tumor cells, bacteria, and circulating platelets/neutrophils of healthy hosts. But there were no data on "sick" hosts to help substantiate any potential GP1 based medical applications. Thus, we adopted similar GP1R binding protocols to further study binding of GP1 in different biological samples (including different blood samples of hosts in sickness and in health, cancer cell lines, and trigeminal ganglia culture of infected hosts treated with and without GP1) and determine if any binding patterns might have any associations with different health conditions. The overall preliminary results appear to show certain GP1R + binding patterns in certain blood components (especially neutrophils) have potential correlations with certain health conditions of hosts at sampling times, indicating potential GP1R applications for diagnostic purposes. Findings of different GP1R binding patterns in different cancer cell lines, whole blood samples and trigeminal ganglia culture of experimental mice infected with HSV-1 virus (might cause neuropathy) within a week post-infection, and blood samples of GP1-treated mouse survivors on day 21 post-infection provided preliminary evidence of potential GP1-led tumor cell-specific cell death and treatment efficacy for greater survival.

Widespread and dynamic expression of granzyme C by skin-resident antiviral T cells.

After recognition of cognate antigen (Ag), effector CD8+ T cells secrete serine proteases called granzymes in conjunction with perforin, allowing granzymes to enter and kill target cells. While the roles for some granzymes during antiviral immune responses are well characterized, the function of others, such as granzyme C and its human ortholog granzyme H, is still unclear. Granzyme C is constitutively expressed by mature, cytolytic innate lymphoid 1 cells (ILC1s). Whether other antiviral effector cells also produce granzyme C and whether it is continually expressed or responsive to the environment is unknown. To explore this, we analyzed granzyme C expression in different murine skin-resident antiviral lymphocytes. At steady-state, dendritic epidermal T cells (DETCs) expressed granzyme C while dermal γδ T cells did not. CD8+ tissue-resident memory T cells (TRM) generated in response to cutaneous viral infection with the poxvirus vaccinia virus (VACV) also expressed granzyme C. Both DETCs and virus-specific CD8+ TRM upregulated granzyme C upon local VACV infection. Continual Ag exposure was not required for maintained TRM expression of granzyme C, although re-encounter with cognate Ag boosted expression. Additionally, IL-15 treatment increased granzyme C expression in both DETCs and TRM. Together, our data demonstrate that granzyme C is widely expressed by antiviral T cells in the skin and that expression is responsive to both environmental stimuli and TCR engagement. These data suggest that granzyme C may have functions other than killing in tissue-resident lymphocytes.

Relevance of tissue-resident memory CD8 T cells in the onset of Parkinson's disease and examination of its possible etiologies: infectious or autoimmune?

Tissue-resident memory CD8 T cells are responsible for local immune surveillance in different tissues, including the brain. They constitute the first line of defense against pathogens and cancer cells and play a role in autoimmunity. A recently published study demonstrated that CD8 T cells with markers of residency containing distinct granzymes and interferon-γ infiltrate the parenchyma of the substantia nigra and contact dopaminergic neurons in an early premotor stage of Parkinson's disease. This infiltration precedes α-synuclein aggregation and neuronal loss in the substantia nigra, suggesting a relevant role for CD8 T cells in the onset of the disease. To date, the nature of the antigen that initiates the adaptive immune response remains unknown. This review will discuss the role of tissue-resident memory CD8 T cells in brain immune homeostasis and in the onset of Parkinson's disease and other neurological diseases. We also discuss how aging and genetic factors can affect the CD8 T cell immune response and how animal models can be misleading when studying human-related immune response. Finally, we speculate about a possible infectious or autoimmune origin of Parkinson's disease.

Granzymes expression patterns predict immunotherapy response and identify the heterogeneity of CD8+ T cell subsets.

BACKGROUND: Recent studies illustrated the effects of granzymes (GZMs) gene alterations on immunotherapy response of cancer patients. Thus, we aimed to systematically analyze the expression and prognostic value of GZMs for immunotherapy in different cancers, and identified heterogeneity of the GZMs expression-based CD8+ T cell subsets. METHODS: First, we analyzed GZMs expression and prognostic value at pan-cancer level. Meanwhile, we established a GZMs score by using the single-sample gene set enrichment analysis (ssGSEA) algorithm to calculate the enrichment scores (ES) based on a gene set of five GZMs. The potential value of GZMs score for predicting survival and immunotherapy response was evaluated using the tumor immune dysfunction and exclusion (TIDE) and immunophenoscore (IPS) algorithm, and we validated it in immunotherapy cohorts. CellChat, scMetabolism, and SCENIC R packages were used for intercellular communication networks, quantifying metabolism activity, and regulatory network reconstruction, respectively. RESULTS: The GZMs score was significantly associated with IPS, TIDE score. Patients with high GZMs score tended to have higher objective response rates of immunotherapy in melanoma and urothelial carcinoma. GZMs expression-based CD8+ T cell subsets presented heterogeneity in functions, metabolism, intercellular communications, and the tissue-resident memory programs in lung adenocarcinoma (LUAD). The transcription factors RUNX3 and ETS1, which may regulate the expression of GZMs, was found to be positively correlated with the tissue-resident memory T cells-related marker genes. CONCLUSIONS: The higher GZMs score may indicate better response and overall survival (OS) outcome for immunotherapy in melanoma and urothelial carcinoma but worse OS in renal cell carcinoma (RCC). The GZMs score is a potential prognostic biomarker of diverse cancers. RUNX3 and ETS1 may be the potential targets to regulate the infiltration of GZMs expression-based CD8+ T cell subsets and affect the tissue-resident memory programs in LUAD, which may affect the prognosis of LUAD patients and the response to immunotherapy.

Role of the granzyme family in rheumatoid arthritis: Current Insights and future perspectives.

Rheumatoid arthritis (RA) is a complex autoimmune disease characterized by chronic inflammation that affects synovial tissues of multiple joints. Granzymes (Gzms) are serine proteases that are released into the immune synapse between cytotoxic lymphocytes and target cells. They enter target cells with the help of perforin to induce programmed cell death in inflammatory and tumor cells. Gzms may have a connection with RA. First, increased levels of Gzms have been found in the serum (GzmB), plasma (GzmA, GzmB), synovial fluid (GzmB, GzmM), and synovial tissue (GzmK) of patients with RA. Moreover, Gzms may contribute to inflammation by degrading the extracellular matrix and promoting cytokine release. They are thought to be involved in RA pathogenesis and have the potential to be used as biomarkers for RA diagnosis, although their exact role is yet to be fully elucidated. The purpose of this review was to summarize the current knowledge regarding the possible role of the granzyme family in RA, with the aim of providing a reference for future research on the mechanisms of RA and the development of new therapies.

CD8+ T Cells Promote Pathological Angiogenesis in Ocular Neovascular Disease.

BACKGROUND: CD4+ (cluster of differentation) and CD8+ T cells are increased in the ocular fluids of patients with neovascular retinopathy, yet their role in the disease process is unknown. METHODS: We describe how CD8+ T cells migrate into the retina and contribute to pathological angiogenesis by releasing cytokines and cytotoxic factors. RESULTS: In oxygen-induced retinopathy, flow cytometry revealed the numbers of CD4+ and CD8+ T cells were increased in blood, lymphoid organs, and retina throughout the development of neovascular retinopathy. Interestingly, the depletion of CD8+ T cells but not CD4+ T cells reduced retinal neovascularization and vascular leakage. Using reporter mice expressing gfp (green fluorescence protein) in CD8+ T cells, these cells were localized near neovascular tufts in the retina, confirming that CD8+ T cells contribute to the disease. Furthermore, the adoptive transfer of CD8+ T cells deficient in TNF (tumor necrosis factor), IFNγ (interferon gamma), Prf (perforin), or GzmA/B (granzymes A/B) into immunocompetent Rag1-/- mice revealed that CD8+ T cells mediate retinal vascular disease via these factors, with TNF influencing all aspects of vascular pathology. The pathway by which CD8+ T cells migrate into the retina was identified as CXCR3 (C-X-C motif chemokine receptor 3) with the CXCR3 blockade reducing the number of CD8+ T cells within the retina and retinal vascular disease. CONCLUSIONS: We discovered that CXCR3 is central to the migration of CD8+ T cells into the retina as the CXCR3 blockade reduced the number of CD8+ T cells within the retina and vasculopathy. This research identified an unappreciated role for CD8+ T cells in retinal inflammation and vascular disease. Reducing CD8+ T cells via their inflammatory and recruitment pathways is a potential treatment for neovascular retinopathies.

Super-killer CTLs are generated by single gene deletion of Bach2.

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8+ T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8+ T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer.

Noncytotoxic Roles of Granzymes in Health and Disease.

Granzymes are serine proteases previously believed to play exclusive and somewhat redundant roles in lymphocyte-mediated target cell death. However, recent studies have challenged this paradigm. Distinct substrate profiles and functions have since emerged for each granzyme while their dysregulated proteolytic activities have been linked to diverse pathologies.

Anti-inflammatory and immune-mediated therapy for a case of febrile infection-related epilepsy syndrome with rapid recurrence.

Febrile infection-related epilepsy syndrome (FIRES) is a disease of unknown etiology, characterized by refractory frequent focal seizures, which require prolonged intensive care. We successfully treated a boy with FIRES with anti-inflammatory and immunosuppressive therapy. This case suggests that an autoimmune mechanism may play a role in the development of FIRES.

Effective Natural Killer Cell Degranulation Is an Essential Key in COVID-19 Evolution.

NK degranulation plays an important role in the cytotoxic activity of innate immunity in the clearance of intracellular infections and is an important factor in the outcome of the disease. This work has studied NK degranulation and innate immunological profiles and functionalities in COVID-19 patients and its association with the severity of the disease. A prospective observational study with 99 COVID-19 patients was conducted. Patients were grouped according to hospital requirements and severity. Innate immune cell subpopulations and functionalities were analyzed. The profile and functionality of innate immune cells differ between healthy controls and severe patients; CD56dim NK cells increased and MAIT cells and NK degranulation rates decreased in the COVID-19 subjects. Higher degranulation rates were observed in the non-severe patients and in the healthy controls compared to the severe patients. Benign forms of the disease had a higher granzymeA/granzymeB ratio than complex forms. In a multivariate analysis, the degranulation capacity resulted in a protective factor against severe forms of the disease (OR: 0.86), whereas the permanent expression of NKG2D in NKT cells was an independent risk factor (OR: 3.81; AUC: 0.84). In conclusion, a prompt and efficient degranulation functionality in the early stages of infection could be used as a tool to identify patients who will have a better evolution.

All About (NK Cell-Mediated) Death in Two Acts and an Unexpected Encore: Initiation, Execution and Activation of Adaptive Immunity.

NK cells are key mediators of immune cell-mediated cytotoxicity toward infected and transformed cells, being one of the main executors of cell death in the immune system. NK cells recognize target cells through an array of inhibitory and activating receptors for endogenous or exogenous pathogen-derived ligands, which together with adhesion molecules form a structure known as immunological synapse that regulates NK cell effector functions. The main and best characterized mechanisms involved in NK cell-mediated cytotoxicity are the granule exocytosis pathway (perforin/granzymes) and the expression of death ligands. These pathways are recognized as activators of different cell death programmes on the target cells leading to their destruction. However, most studies analyzing these pathways have used pure recombinant or native proteins instead of intact NK cells and, thus, extrapolation of the results to NK cell-mediated cell death might be difficult. Specially, since the activation of granule exocytosis and/or death ligands during NK cell-mediated elimination of target cells might be influenced by the stimulus received from target cells and other microenvironment components, which might affect the cell death pathways activated on target cells. Here we will review and discuss the available experimental evidence on how NK cells kill target cells, with a special focus on the different cell death modalities that have been found to be activated during NK cell-mediated cytotoxicity; including apoptosis and more inflammatory pathways like necroptosis and pyroptosis. In light of this new evidence, we will develop the new concept of cell death induced by NK cells as a new regulatory mechanism linking innate immune response with the activation of tumour adaptive T cell responses, which might be the initiating stimulus that trigger the cancer-immunity cycle. The use of the different cell death pathways and the modulation of the tumour cell molecular machinery regulating them might affect not only tumour cell elimination by NK cells but, in addition, the generation of T cell responses against the tumour that would contribute to efficient tumour elimination and generate cancer immune memory preventing potential recurrences.

Granzymes-Their Role in Colorectal Cancer.

Colorectal cancer (CRC) is among the most common malignancies worldwide. CRC is considered a heterogeneous disease due to various clinical symptoms, biological behaviours, and a variety of mutations. A number of studies demonstrate that as many as 50% of CRC patients have distant metastases at the time of diagnosis. However, despite the fact that social and medical awareness of CRC has increased in recent years and screening programmes have expanded, there is still an urgent need to find new diagnostic tools for early detection of CRC. The effectiveness of the currently used classical tumour markers in CRC diagnostics is very limited. Therefore, new proteins that play an important role in the formation and progression of CRC are being sought. A number of recent studies show the potential significance of granzymes (GZMs) in carcinogenesis. These proteins are released by cytotoxic lymphocytes, which protect the body against viral infection as well specific signalling pathways that ultimately lead to cell death. Some studies suggest a link between GZMs, particularly the expression of Granzyme A, and inflammation. This paper summarises the role of GZMs in CRC pathogenesis through their involvement in the inflammatory process. Therefore, it seems that GZMs could become the focus of research into new CRC biomarkers.

Early Response of CD8+ T Cells in COVID-19 Patients.

Healthy and controlled immune response in COVID-19 is crucial for mild forms of the disease. Although CD8+ T cells play important role in this response, there is still a lack of studies showing the gene expression profiles in those cells at the beginning of the disease as potential predictors of more severe forms after the first week. We investigated a proportion of different subpopulations of CD8+ T cells and their gene expression patterns for cytotoxic proteins (perforin-1 (PRF1), granulysin (GNLY), granzyme B (GZMB), granzyme A (GZMA), granzyme K (GZMK)), cytokine interferon-γ (IFN-γ), and apoptotic protein Fas ligand (FASL) in CD8+ T cells from peripheral blood in first weeks of SARS-CoV-2 infection. Sixteen COVID-19 patients and nine healthy controls were included. The absolute counts of total lymphocytes (p = 0.007), CD3+ (p = 0.05), and CD8+ T cells (p = 0.01) in COVID-19 patients were significantly decreased compared to healthy controls. In COVID-19 patients in CD8+ T cell compartment, we observed lower frequency effector memory 1 (EM1) (p = 0.06) and effector memory 4 (EM4) (p < 0.001) CD8+ T cells. Higher mRNA expression of PRF1 (p = 0.05) and lower mRNA expression of FASL (p = 0.05) at the fifth day of the disease were found in COVID-19 patients compared to healthy controls. mRNA expression of PRF1 (p < 0.001) and IFN-γ (p < 0.001) was significantly downregulated in the first week of disease in COVID-19 patients who progressed to moderate and severe forms after the first week, compared to patients with mild symptoms during the entire disease course. GZMK (p < 0.01) and FASL (p < 0.01) mRNA expression was downregulated in all COVID-19 patients compared to healthy controls. Our results can lead to a better understanding of the inappropriate immune response of CD8+ T cells in SARS-CoV2 with the faster progression of the disease.

Oxidative and Non-Oxidative Antimicrobial Activities of the Granzymes.

Cell-mediated cytotoxicity is an essential immune defense mechanism to fight against viral, bacterial or parasitic infections. Upon recognition of an infected target cell, killer lymphocytes form an immunological synapse to release the content of their cytotoxic granules. Cytotoxic granules of humans contain two membrane-disrupting proteins, perforin and granulysin, as well as a homologous family of five death-inducing serine proteases, the granzymes. The granzymes, after delivery into infected host cells by the membrane disrupting proteins, may contribute to the clearance of microbial pathogens through different mechanisms. The granzymes can induce host cell apoptosis, which deprives intracellular pathogens of their protective niche, therefore limiting their replication. However, many obligate intracellular pathogens have evolved mechanisms to inhibit programed cells death. To overcome these limitations, the granzymes can exert non-cytolytic antimicrobial activities by directly degrading microbial substrates or hijacked host proteins crucial for the replication or survival of the pathogens. The granzymes may also attack factors that mediate microbial virulence, therefore directly affecting their pathogenicity. Many mechanisms applied by the granzymes to eliminate infected cells and microbial pathogens rely on the induction of reactive oxygen species. These reactive oxygen species may be directly cytotoxic or enhance death programs triggered by the granzymes. Here, in the light of the latest advances, we review the antimicrobial activities of the granzymes in regards to their cytolytic and non-cytolytic activities to inhibit pathogen replication and invasion. We also discuss how reactive oxygen species contribute to the various antimicrobial mechanisms exerted by the granzymes.