Eswari Marri - Academia.edu (original) (raw)

Papers by Eswari Marri

Secretion levels for GROα (A), IL-6 (B), IL-6 Ra (C) and IL-8 (D), were measured by a Ma... more Secretion levels for GROα (A), IL-6 (B), IL-6 Ra (C) and IL-8 (D), were measured by a MagPix Milliplex kit (EMD Millipore) rhSP-A treatments (10 and 20 μg/ml) caused an increase in the expression of GROα and IL-8 compared to the untreated cells, but did not have a profound effect on IL-6 and IL-6Ra. rhSP-D treated cells (10 and 20 μg/ml) appeared to express higher levels of GROα, IL-6 and IL-8 at 12 and 24 h compared to the untreated cells. ULTR cells treated with rhSP-D (10 and 20 μg/ml) expressed higher levels of IL-6 Ra after 24h. (*p<0.05, **p<0.01, ***p<0.001). Letter denotes values significantly different from each other as indicated by the horizontal and vertical lines (p<0.05).</p

αvβ8 integrin, a key activator of transforming growth factor β (TGF-β), inhibits anti-tumor immun... more αvβ8 integrin, a key activator of transforming growth factor β (TGF-β), inhibits anti-tumor immunity. We show that a potent blocking monoclonal antibody against αvβ8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma, mammary cancer, colon cancer, and prostate cancer, especially when combined with other immunomodulators or radiotherapy. αvβ8 is expressed at the highest levels in CD4+CD25+ T cells in tumors, and specific deletion of β8 from T cells is as effective as ADWA-11 in suppressing tumor growth. ADWA-11 increases expression of a suite of genes in tumor-infiltrating CD8+ T cells normally inhibited by TGF-β and involved in tumor cell killing, including granzyme B and interferon-γ. The in vitro cytotoxic effect of tumor CD8 T cells is inhibited by CD4+CD25+ cells, and this suppressive effect is blocked by ADWA-11. These findings solidify αvβ8 integrin as a promising target for cancer immunotherapy

Total term decidual proteins (30 µg each sample) were blotted and probed with mouse mono... more Total term decidual proteins (30 µg each sample) were blotted and probed with mouse monoclonal antibody to SP-D or polyclonal antibody to β-actin; A) representative images (n = 5/group) B) Relative quantity of SP-D in each group was obtained by Labscan and 1D ImageQuant analysis tools using β-actin as internal reference control for normalization. Bars are mean ±SEM; *p<0.05.</p

The 5 µm thin tissue sections a) placenta or c) decidua were probed with monoclonal anti... more The 5 µm thin tissue sections a) placenta or c) decidua were probed with monoclonal antibody to SP-A. Anti-MBL monoclonal antibody was applied to b) placenta or d) decidua. e) Negative controls for the respective tissues. The tissue sections were counterstained with hematoxylin and mounted with DPX. Representative images are 400× original magnification. f) The relative intensity of SP-A and MBL staining was quantitated by NIS elements version 4.1 software analysis tools. Bars are mean ±SEM, *p<0.05 and **p>0.05.</p

Details of primer pair sequences, annealing temperature and product length for amplifica... more Details of primer pair sequences, annealing temperature and product length for amplification of each transcript.</p

Schematic diagram of plausible functions of SP-A and SP-D at the term feto-maternal inte... more Schematic diagram of plausible functions of SP-A and SP-D at the term feto-maternal interface during labor.</p

(n = 5/group). A) Semi-quantitation of the transcripts assessed by real time RT-PCR betw... more (n = 5/group). A) Semi-quantitation of the transcripts assessed by real time RT-PCR between two groups of placenta from caesarean section with no labor (‘NLc’) and placenta from normal vaginal delivery with spontaneous labor (‘SLv’). The experiments were carried out 3 times for each transcript and relative quantity was calculated by 2−ΔΔCt method after normalizing with 18S rRNA of the respective sample. The relative fold expression presented is compared to controls; error bars represent ±SEM for each transcript. B) Western blot analysis of SP-A, SP-D and MBL in total placental protein (30 µg) of both groups (‘NLc’ vs ‘SLv’; n = 5/group). The blots were probed with mouse monoclonal antibody to SP-A (i), SP-D (ii), MBL (iii), and rabbit polyclonal antibody to β-actin for normalization (iv). C) Densitometric analysis of blots for SP-A, SP-D and MBL proteins using Labscan and ImageQuant TL analysis tools (GE Healthcare, USA) and presented as mean ±SEM; i) SP-A, ii) SP-D and iii) MBL. Statistical analysis was performed with one way ANOVA at p<0.05.</p

Cell movements (n = 25 cells, from a single experiment) were observed either without tre... more Cell movements (n = 25 cells, from a single experiment) were observed either without treatment or with treatment with surfactant proteins (10 μg/ml). Both treatments led to a significant increase in velocity, distance travelled and their displacement from their initial position compared to untreated cells. rhSP-A treatment led to a significant increase in velocity and distance compared to rhSP-D treated cells. There was no apparent significance in displacement in between treatments.</p

rhSP-D treatments resulted in an increase of SP-A1, SP-A2 and SP-D mRNA expression at 6 ... more rhSP-D treatments resulted in an increase of SP-A1, SP-A2 and SP-D mRNA expression at 6 h at 5 and 10 μg/ml. The effect was inverted at 12 h, with rhSP-D leading to a decrease in the expression of the above genes (n = 3).</p

Primer sequences of human gene targets used for qPCR experiments.</p

Untreated cells (A) failed to close the gap even after 48 h (D), whereas the cells treat... more Untreated cells (A) failed to close the gap even after 48 h (D), whereas the cells treated with rhSP-A (B) closed the gap after 17 h (E) while those treated with rhSP-D (C) after 25 h (F). Dotted rectangular annotates the area covered by treated ULTRs. Gap that was less than 3 cells apart was considered as closed. x 10 magnification.</p

Nuclear staining using phalloidin 488 for SP-A (B) and SP-D (E). Merged images for SP-A ... more Nuclear staining using phalloidin 488 for SP-A (B) and SP-D (E). Merged images for SP-A (C) and SP-D (F). x 40 magnification. (G) and (H): Representative cells immunostained for SP-A and SP-D respectively. Merged image (I) showing the comparison of the fluorescence intensity of the cells between SP-A (red) and SP-D (black). SP-D immunostained cells appeared to have higher intensity compared to SP-A immunostained cells.</p

rhSP-A led to an increase of CX43 mRNA expression after 4 h at a concentration of 10 and... more rhSP-A led to an increase of CX43 mRNA expression after 4 h at a concentration of 10 and 20 μg/ml, effect that seemed to disappear after 6 h, and of OXTR after 6 h at concentrations of 10 and 20 μg/ml. rhSP-D resulted in an increase of CX43 transcript at all concentrations, and of OXTR transcript after 6 h at a concentration of 5 μg/ml (n = 3). Letter denotes values significantly different from each other as indicated by the horizontal and vertical lines (p<0.05).</p

Cells were seeded at a specific density and were grown in a collagen matrix to mimic a 3... more Cells were seeded at a specific density and were grown in a collagen matrix to mimic a 3D milieu. rhSP-A and rhSP-D significantly induced a similar contractility of ULTR cells compared to untreated cells, at 3 and 24 h (A), (*p<0.05, **p<0.01, ***p<0.001). There was no significance in between treatments or time points. Representative collagen discs of 3 h treatment (B; NS: no supplement). Insert: image of ULTRs grown within the collagen matrix (n = 4).</p

doi: 10.3389/fimmu.2012.00131 An insight into the diverse roles of surfactant proteins, SP-A and ... more doi: 10.3389/fimmu.2012.00131 An insight into the diverse roles of surfactant proteins, SP-A and SP-D in innate and adaptive immunity

Surfactant protein D (SP-D) is a carbohydrate/charged pattern recognition molecule of the innate ... more Surfactant protein D (SP-D) is a carbohydrate/charged pattern recognition molecule of the innate immune system. By virtue of its ability to recognize an array of carbohydrate patterns on the surface of a range of pathogens, SP-D can bring about opsonisation, enhanced phagocytosis and killing of a diverse range of viruses, bacteria and fungi. In addition to antimicrobial functions, which also includes bacteriostatic and fungistatic properties SP-D has also been shown to bind allergens derived from a number of sources including house dust mite, Aspergilllus fumigatus and pollen grains. SP-D allergen interaction leads to inhibition of specific IgE binding and subsequent downregulation of histamine release from sensitized basophils and mast cells. Thus, a number of murine models of pulmonary hypersensitivity and allergic asthma induced by ovalbumin, house dust mite and Aspergillus fumigatus allergens/antigens have been tested for the ability of SP-D to dampen allergic symptoms on the immunological parameters. In general, treatment of allergic models with a recombinant fragment of human SP-D (rh SP-D; composed of trimeric, neck and carbohydrate recognition domain) has been shown to cause downregualtion of specific IgE synthesis, pulmonary and peripheral eosinophilia and airway hyper reactivity, and Th2→Th1 polarisation. However, therapeutic alleviation of eosinophilia by rh SP-D treatment became evident when SP-D gene deficient mice were found to be hypereosinophilic In fact, rhSP-D binds well to eosinophils derived from allergic patients and induces apoptosis without affecting eosinophils derived from healthy individuals or nonactivated/non-sensitized eosinophils. Proteomic analysis of rh SP-D treated eosinophillic cell line that revealed that apoptosis induction takes place via p53 pathway. In this thesis, proteomic signatures were replicated using a leukemic cell line AML14.3D10 via qPCR analysis by identifying targets from a spectrum of genes, which were either upregulated or downregulated. It appears that in spite of induction of apoptosis by rh SP-D, different cells respond differentially at molecular levels (Chapter 3). Sensing that SP-D can induce apoptosis in altered or transformed cells; the effect of SP-D gene expression within pancreatic cancer cells was also investigated. The experiments confirmed p53 pathway dependence for suppression of cancer. Interestingly, factors responsible for metastasis for cancer are also downregulated by endogenous overexpression of SP-D, as validated by wound healing assay. We conclude that SP-D is a general immunosurveillance molecule, which is involved in the clearance of altered and transformed cells (Chapter 4). Chapter 5 shows a direct interaction between DC-SIGN and rh SP-D that inhibits DC-SIGN interaction of allergens and HIV-1, tow common ligands for SP-D and DC-SIGN. Using transfected human embryonic kidney (HEK) cells expressing surface DC-SIGN, we found that pre-treatment of these cells with rhSP-D suppressed DC-SIGN mediated transmission of HIV-1to co-cultured PBMCs. The effect of rhSP-D-DC-SIGN In conclusion, this

Cell Reports, 2021

Integrin avb8 on T cells suppresses anti-tumor immunity in multiple models and is a promising tar... more Integrin avb8 on T cells suppresses anti-tumor immunity in multiple models and is a promising target for tumor immunotherapy Graphical abstract Highlights d ITGB8 on CD4+/CD25+ T cells activates latent TGF-b, causing tumor immunosuppression d Anti-ITGB8 therapy elicits tumor regression and durable antitumor immunity d CD8+T cells are essential for anti-tumor activity of anti-ITGB8 d Anti-ITGB8 synergizes with multiple immunomodulators in multiple tumor types

SSRN Electronic Journal, 2020

The αvβ8 integrin is a key activator of transforming growth factor β(TGFβ), which has been shown ... more The αvβ8 integrin is a key activator of transforming growth factor β(TGFβ), which has been shown to inhibit anti-tumor immunity. Previous work has suggested that αvβ8 on tumor cells could modulate tumor growth and responses to immune checkpoint blockade. We now show that a potent blocking monoclonal antibody against avb8 (ADWA-11) causes growth suppression or complete regression in syngeneic models of squamous cell carcinoma (CCK168), mammary cancer (EMT-6), colon cancer (CT26), and prostate cancer (TRAMPC2), especially when it is combined with other immunomodulators (anti-PD-1, anti-CTLA-4 or 4-1BB) or radiotherapy. αvβ8 is expressed on tumor cells in some of these models, but tumor cell expression of avb8 is not essential for the beneficial effects of ADWA-11 therapy. αvβ8 is consistently expressed at highest levels on CD4+CD25+ T cells within tumors, and specific deletion of Itgb8 from T cells is as effective as ADWA-11 in suppressing tumor growth. Treatment with ADWA-11 increases expression of a suite of genes in tumor infiltrating CD8+ T cells that are normally inhibited by TGFβ and are involved in tumor cell killing, including Granzyme B and Interferon-g. These findings solidify αvβ8 integrin as a promising target for cancer immunotherapy, even for tumors that do not express this integrin.

Journal for ImmunoTherapy of Cancer, 2019

Background: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, bu... more Background: Checkpoint blockade immunotherapy has improved metastatic cancer patient survival, but response rates remain low. There is an unmet need to identify mechanisms and tools to circumvent resistance. In human patients, responses to checkpoint blockade therapy correlate with tumor mutation load, and intrinsic resistance associates with pre-treatment signatures of epithelial mesenchymal transition (EMT), immunosuppression, macrophage chemotaxis and TGFβ signaling. Methods: To facilitate studies on mechanisms of squamous cell carcinoma (SCC) evasion of checkpoint blockade immunotherapy, we sought to develop a novel panel of murine syngeneic SCC lines reflecting the heterogeneity of human cancer and its responses to immunotherapy. We characterized six Kras-driven cutaneous SCC lines with a range of mutation loads. Following implantation into syngeneic FVB mice, we examined multiple tumor responses to α-PD-1, α-TGFβ or combinatorial therapy, including tumor growth rate and regression, tumor immune cell composition, acquired tumor immunity, and the role of cytotoxic T cells and Tregs in immunotherapy responses. Results: We show that α-PD-1 therapy is ineffective in establishing complete regression (CR) of tumors in all six SCC lines, but causes partial tumor growth inhibition of two lines with the highest mutations loads, CCK168 and CCK169. α-TGFβ monotherapy results in 20% CR and 10% CR of established CCK168 and CCK169 tumors respectively, together with acquisition of long-term anti-tumor immunity. α-PD-1 synergizes with α-TGFβ, increasing CR rates to 60% (CCK168) and 20% (CCK169). α-PD-1 therapy enhances CD4 + Treg/CD4 + Th ratios and increases tumor cell pSmad3 expression in CCK168 SCCs, whereas α-TGFβ antibody administration attenuates these effects. We show that α-TGFβ acts in part through suppressing immunosuppressive Tregs induced by α-PD-1, that limit the anti-tumor activity of α-PD-1 monotherapy. Additionally, in vitro and in vivo, α-TGFβ acts directly on the tumor cell to attenuate EMT, to activate a program of gene expression that stimulates immuno-surveillance, including up regulation of genes encoding the tumor cell antigen presentation machinery.

Primer sequences of human gene targets used for qPCR experiments.</p

Cell Reports, 2021

SSRN Electronic Journal, 2020

Journal for ImmunoTherapy of Cancer, 2019